http://2014.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=250&target=Jordyevan&year=&month=2014.igem.org - User contributions [en]2024-03-29T16:01:40ZFrom 2014.igem.orgMediaWiki 1.16.5http://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-18T01:44:18Z<p>Jordyevan: </p>
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<br><h2>Riboregulator Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
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<br><br><b>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:</b><br><br />
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<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
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<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
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<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
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<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
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<td scope="row">Lock 3c <a href="http://parts.igem.org/Part:BBa_J23031">BBa_J23031 </a> Key 3c<a href="http://parts.igem.org/Part:BBa_J23008">BBa_J23008 </a></td> <br />
<td>iGEM 2006_Berkeley</td> <br />
<td>No</td> <br />
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<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
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<div class='content_1'><h3>Riboregulator Results</h3><br />
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<a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br><img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
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<h5 style="font-size: 13px">Figure 1. Fluorescence (F)/OD600 measurements of riboregulator pairs after arabinose induction and their corresponding controls. </h5><br />
<h6 style= "font-size: 13px"> All samples were inoculated in M9 minimal salt medium overnight in no or various arabinose concentration (%w/v). The samples were diluted around 10 fold the next day. Measurements were made the samples reached around the mid-log phase (OD600 = 0.3 to 0.5). Graphs depict the triplicate mean + standard deviation. (A) Schematic diagram of the genetic context of the experiment. Note that the diagram generalized the CR and TA sequences. (B) Measurement for Lock 1 (<a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010</a>) and Key 1 (<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a>) cognate pair. (C) Measurement for Lock 3(<a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080</a>) and Key 3 (<a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086</a>) cognate pair. (D) Measurement for Medium lock (<a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a>) and Key for medium lock (<a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032</a>) cognate pair. (E) Measurement for Lock 3c (<a href="http://parts.igem.org/Part:BBa_J23031">BBa_J23031</a>) and Key 3c (<a href="http://parts.igem.org/Part:BBa_J23008">BBa_J23008</a>). </h6><br />
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<p><br />
To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the RBS. The RBS sequence also had to be different for some of the riboregulator system because the cr-repressing sequence depends on the RBS sequence; In order to repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter (<a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a>) to drive the expression of the cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>). The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (Isaacs et al., 2004) (Figure 1. A). <br />
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<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). Almost full repression was observed for the three cognate pairs. For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). One possible reason could be that the RBS sequence that we used for the controls of Lock 3c Key 3c was incorrect. For Lock3c the target RBS sequence was not mentioned. It seemed like a variation of <a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034 </a> with shorter 3’ end. In order to build the construct type 2 and 3 (Figure 1. A), the RBS sequence had to be deduced from the Lock 3c sequence. From the Lock 3c sequence, we have used a part of sequence that resembled the RBS (<a href="http://parts.igem.org/Part:BBa_B0034">BBa_B0034 </a>). The RBS sequence used may have been too short to be functional. Therefore, no fluorescence is observed when cis-repressing sequence is not present. On the other hand, fluorescence can be observed when cis-repressing sequence is present because firstly, the RBS is sequence is correct, and secondly because the cis-repressing sequence failed to repress the translation. <br><br> </p><br />
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After repression, the system needs to be activated when taRNA is expressed. After the addition of arabinose, taRNA is expressed. Out of the three cognate riboregulator pairs that got repressed, only two showed significant increase after arabinose induction. Lock 1- Key 1 cognate paired showed around 13-fold increase for both 1% and 2.5% (%w/v) arabinose induction. Lock 3- Key 3 cognate paired showed around 1.5 and 3 fold increase for 1% and 2.5% of arabinose induction respectively. Lock 1-Key 1 and Lock 3- Key 3 behaved differently for different concentration of arabinose induction. Full induction was observed at 1% arabinose for Lock 1- Key 1 cognate pairs while full induction for Lock 3- Key 3 was observed at 2.5% arabinose. Statistically, no significant fold increase could be observed for Lock m- Key m cognate pair. </p><br />
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<div class='content_1'><h3>Discussion</h3><br />
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<p><i>The fold increase for Lock 1- Key 1 is lower than that of riboregulator pair mentioned in the Isaacs et al.’s paper </i><br><br><br />
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iGEM 2005_Berekely, when they first introduced the riboregulator system to the iGEM community, they mentioned in the Part Registry page that Lock 1 and Key 1 are “Biobricked version of Isaacs’ riboregulator” crR12 and taR12 respectively. We can therefore expect the Lock 1 and Key 1 fold increase after induction to be similar to what Isaacs et al. have observed and mentioned in the paper. Isaacs et al. mention that for crR12 and taR12 cognate pair, they have observed 19-fold increase (Isaacs et al., 2004), our results showed only showed around 13 fold increase. One possibility for the deviation could be because the Lock 1 and Key 1 sequences are not 100% match with crR12 and taR12. Because scars are introduced in 5’ and 3’ end of a Biobricked parts, iGEM 2005_Berekeley had to shorten the original 5’ and 3’ end of the crR12 and taR12. This change actually changes the crRNA and taRNA sequence and therefore could result to the deviated results. Another possibility is simply because the genetic context that we did our characterization was different from what Isaac et al. used to characterize their riboregulator system. If this is the case, we can at least see that the system is not very modular: changing the genetic context can change the fold increase. <br><br><br />
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<i>The fluorescence after induction is still low compared to that of unrepressed controls. </i><br><br><br />
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Although we saw significant fold increase for two riboregulator systems that we have characterize, compared to the fluorescence of unrepressed controls, the fluorescence is very low. For Lock 1-Key 1 riboeregulator system, the fluorescence after induction only correspond to around 0.4% of the that of the unrepressed control. For the Lock 3-Key3 system, the value was around 0.3%. The lower expression partly is because of the lower mRNA levels. After the introduction of the cis-repressing sequence the mRNA level was 40% of that of the controls (Isaacs et al., 2004). Another reason for low fluorescence after induction could have resulted because of our genetic context. We have used a relatively strong constitutive promoter (<a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102</a>) to express the crRNA and a relatively weak arabinose inducible promoter (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>). This could have caused imbalance of crRNA and taRNA levels. We could have had lower taRNA level and therefore failed to fully activate the riboregulator system. Further investigation is required. Simply changing the arabinose inducible promoter to a strong promoter can tell us whether this is the case. <br><br><br />
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<i>Different Lock-Key cognate pairs behaved differently to different arabinose concentration.</i><br><br><br />
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Lock 1- Key 1 riboregualtor cognate pair was fully induced and leveled off at 1% arabinose concentration. For Lock 3- Key 3 pair, the full induction was observed at 2.5% arabinose concentration. We did not conduct more investigation to understand the difference in the response. Further investigation on how changing the riboregulator sequence can change the sensitivity of the system could be an interesting topic. <br />
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<div class='content_1'><h3>Methods</h3><br />
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<p><i>Fluorometry </i><br><br><br />
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Triplicate of each sample were inoculated overnight in a deep-well 96 well plate. M9 minimal salt solution was used for the inoculation because it gives low background fluorescence. 1% and 2.5% (w/v) arabinose concentration was used for overnight induction. Samples were diluted around 10-fold the next day and regrown to mid-log phase (OD600=0.3 ~ 0.6). 200µl of sample were drawn out from the deep-well plate and plated on a clear round bottom plate for measurement. For each sample, OD595 and Fluorescence was measured using EnVision Multilabel Reader. The excitation wavelength was 485/14nm and the emission wavelength was 535/25nm. Conversion factor for OD595 to OD600 was obtained by calculating the slope of the OD600 v.s. OD595 graph. The conversion factor, 1.24 was multiplied to OD595 reading to covert the measurements to OD600. Background fluorescence was subtracted for each fluorescence measurement by making another standard Fluorescence v.s. OD600 graph. DH10B with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used to produce the standard curve. The corrected fluorescence was then divided by the corresponding OD600. Finally the average and standard deviation were calculated. <br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
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<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD. The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can activate the P<sub>BAD</sub> promoter (Schleif, 2010).</p><br />
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<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
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<p> <br />
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There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter responded to the arabinose induction. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge results show that it was an “on-or-off” response. We wanted to analyze these problems so that the part could be more reliable for other users. Cambridge cited a paper that mentioned that variation in response could have resulted from cell strain variation Khelbnikov, 2001)</p><br />
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<div class='content_1'><h3>Results</h3><br />
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<p><u>All-or-none response observed for individual cells</u>.<br>Flow cytometry can measure the fluorescence of individual cells. The individual measurement of cell fluorescence can be plotted in a forward scatter (FSC) intensity value versus fluorescence graph. An arbitrary vertical line divided the region of the graph with low fluorescence (Q3) and high fluorescence (Q4). <br><br><br />
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For the negative control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, we see cells distributed in Q3 while absent in Q4. Converse was true for the positive control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (GFP generator regulated by <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>). We see most cells distributed in Q4. For our experimental sample, (pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) similar trend could observed for three strains. At no arabinose induction, we see most of the cells in Q3. The distribution of cells shifted to Q4 when arabinose was added to the medium. We also see that the cells remained in Q3 even after arabinose induction. The bimodal distribution indicate that some cells got induced while other cells were in repressed state. <br />
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<img style="width:80%; display: block;<br />
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<h5 style="font-size: 13px">Figure 1. Forward scatter intensity (FSC) versus GFP graphs for samples with P<sub>BAD</sub> promoter regulating GFP generator. </h5><br />
<h6 style= "font-size: 13px"> All samples were inoculated in M9 minimal salt medium overnight in various arabinose concentrations (%w/v). The samples were diluted around 10 fold the next day. Sample were fixed and the fluorescence was measured using flow cytometer. The graphs were plotted for the control constructs, pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (-) and pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (<a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>) in the absence of arabinose. FSC versus GFP graphs for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>(<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>) in 0, 0.2 and 1.0% arabinose concentration were plotted. Each set of graphs were obtained for three different cell strains, DH10B, DH5α and BW25113. </h6> <br><br><br />
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<p>The distribution can also give us some idea of P<sub>BAD</sub> promoter leakage in different cells strain. If the data points are on or near the boundary between Q3 and Q4, we know that the promoter is leaky. The results indicate that the promoter is relatively more leaky in DH10B compared to other cell strains. <br><br><br />
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The percentages of cells in each Q3 and Q4 are highlighted in Figure 2. After addition of arabinose, we clearly see more percentage in Q4. Around 80% of the cells for DH10B and BW25113 are in Q4. Relatively lower percentage of cells in Q4 is observed for DH5α. Only around 60 to 70% of cells were in Q4 after arabinose induction. The shift in the distribution from Q3 to Q4 also corresponded to the increase of RPU across different arabinose concentration. At 0% arabinose concentration, for all three strain, higher percentage of cells are in Q3 and therefore the RPU is low. After arabinose induction, more cells are in Q4 and RPU is high. The percentage of cells in Q4 levels off for arabinose concentration that we have tested. The trend was observed for RPU measurement. <br><br><br />
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We can also conclude that even after maximum period of induction (overnight induction) of arabinose, we still see some cells uninduced (in Q3). <br><br><br />
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<img style="width:80%; display: block;<br />
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<h5 style="font-size: 13px">Figure 2. The percentage of cells in induced and uninduced state, and RPU across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Q3 and Q4 represent the 3<sup>rd</sup> and 4<sup>th</sup> quadrants of the forward scatter versus GFP curve mentioned in Figure 2. The experimental condition was same as the procedure mentioned in the caption of the Figure 1. The left y-axis is for the percent of cells in Q3 and Q4 while the right y-axis is for RPU. Graphs depict the triplicate mean ± standard deviation. (A) Graph for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in DH10B. (B) Graph for pSBK3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in DH5α. (C) Graph for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in BW25113. </h6> <br><br><br />
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<b><u>RPU of PBAD in different cell strains</u></b><br><br />
In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D graph while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another strain, BW25113, which is commonly used by synthetic biologists. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). <br><br><br />
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<img style="width:80%; display: block;<br />
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<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using flow cytometer. The graph represent triplicate mean ±SD. </h6> <br><br><br />
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We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by other experimental factors such as genetic context. Because RPU is a ratio to fluorescence measurement, the effects caused by these factors can be minimized (See Methods for RPU calculation). <br><br><br />
<br />
We observed clearly a different promoter response among the three cell strains. The all-or-none response was observed for the strains, but the levels of RPU were different. For DH10B, RPU leveled off around 0.45. For BW25113, RPU leveled off around 0.3. For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The RPU was around 0.16 for DH10B which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were below RPU of 0.1. The lowest leakage was observed for DH5α. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to produce a 3-D graph. We, however, had some changes from the genetic context that Groningen 2011 used. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840</a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator). We also used a low copy pSB3K3 plasmid as the backbone instead of the high copy pSB1C3. We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 4. A and B) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 4. Fluorescence and OD600 measurements of DH10B and DH5α induced in different arabinose concentrations. </h5><br />
<h6 style= "font-size: 13px"> Triplicate of DH10B and DH5α samples were inoculated in deep well 96 well plate overnight in M9 minimal salt medium. Arabinose was added to match the final working concentration from 0 to 1.0 % (w/v) with 0.2% increments. Fluorescence and OD600 was measured every two hours for ten hours. (A) Increase of OD600 measurement for DH10B strain in different arabinose concentration. The graph represents triplicate mean ± SD (B) Increase of OD600 measurement for DH5α strain in different arabinose concentration. (C) Fluorescence VS arabinose concentration VS Time 3-D graph for DH10B. Each point represent triplicate mean. (D) Fluorescence VS arabinose concentration VS Time 3-D graph for DH5α. Each point represent triplicate mean. </h6> <br><br><br />
</p><br />
<br />
<p><br />
The OD600 value reflects cell concentration. OD600 for both strain in different arabinose concentration increased exponentially. Growth rate for cells without arabinose induction (0%) was greatest for both DH10B and DH5α. For other concentration of arabinose induction the growth rates were similar for both of the cell strains (Figure 4. A and B). The similar growth rate across the 10 hour period indicates that the cell concentration for samples in different arabinose concentration increased similarly and therefore the cell concentration at each point of measurement was similar across different arabinose concentration. We can therefore assume that differences in the cell concentrations have minimal effect on the different fluorescence levels at different arabinose concentration. <br><br><br />
<br />
Unlike what Groningen 2011 observed in their experiment, (<a href="http://parts.igem.org/Part:BBa_I0500:Experience">Experience page.</a>) we observed all-or-none response for both of the cell strains. For DH10B and DH5α the fluorescence levelled off at 0.2% arabinose concentration. In the process of subtracting autofluorescence, we got some negative corrected fluorescence. These region correspond to the blue areas of the 3-D graphs. <br />
<br />
<br />
</p><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p><i>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter </i><br><br><br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Groningen 2011 team did not provide with OD600 versus time graphs. Therefore it is hard to tell well there was variation in the cell growth under the context of Groningen 2011’s experiment. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a>(<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) showed gradual response to increasing arabinose concentration. <br><br><br />
<br />
<i>Different genetic context could be responsible for the different response of the P<sub>BAD</sub> promoter. </i><br><br><br />
<br />
Due to lack of human-power, unfortunately, we could not produce 3-D graphs for cells with pSB1C3 backbone as controls. Assuming that Groningen 2011 result was valid, we can speculate that difference of the P<sub>BAD</sub> promoter response could have resulted from the different backbone that we have used to characterize the promoter. In fact, Cambridge 2011, who also observed all-or-none response (<a href="http://parts.igem.org/Part:BBa_I0500:Experience">Experience page.</a>) used pSB3K3 as backbone for their constructs. Without proper controls, it is hard to conclude <br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br />
<br />
Based on this equation, the GFP synthesis rate (dF/dt) for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the experiment construct. GFP synthesis rate (dF/dt) for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the GFP synthesis rate/ABS of experiment with the GFP synthesis rate/ABS of the reference promoter.<br />
<br />
<br />
<br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
Based on this equation, the amount of fluorescence [F] for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the experiment construct. Amount of fluorescence [F] for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the fluorescence*growth rate of experiment with the fluorescence*growth rate of the reference promoter.<br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
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<u><b>References</b></u><br><br></p><br />
<p><br />
<br />
Schleif R. <i>AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action.</i> FEMS Microbiol Rev (2010) 1–18.<br><br><br />
<br />
Khelbnikov, A., Datsenko, K., Skaug, T., Wanner, B., & Keasling, J. (2001).<i> Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. </i> Microbiology, 147(12), 3241-3247.<br><br><br />
<br />
J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4<br />
<br />
Isaacs, F., Dwyer, D., Ding, C., Pervouchine, D., Cantor, C., & Collins, J. (2004).<i> Engineered riboregulators enable post-transcriptional control of gene expression. </i> Nature Biotechnology, 841-847.<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-18T01:11:14Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
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<br />
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<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
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<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
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<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
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<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD. The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can activate the P<sub>BAD</sub> promoter (Schleif, 2010).</p><br />
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<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
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<p> <br />
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There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter responded to the arabinose induction. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge results show that it was an “on-or-off” response. We wanted to analyze these problems so that the part could be more reliable for other users. Cambridge cited a paper that mentioned that variation in response could have resulted from cell strain variation Khelbnikov, 2001)</p><br />
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<div class='content_1'><h3>Results</h3><br />
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<p><u>All-or-none response observed for individual cells</u>.<br>Flow cytometry can measure the fluorescence of individual cells. The individual measurement of cell fluorescence can be plotted in a forward scatter (FSC) intensity value versus fluorescence graph. An arbitrary vertical line divided the region of the graph with low fluorescence (Q3) and high fluorescence (Q4). <br><br><br />
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For the negative control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, we see cells distributed in Q3 while absent in Q4. Converse was true for the positive control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (GFP generator regulated by <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>). We see most cells distributed in Q4. For our experimental sample, (pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) similar trend could observed for three strains. At no arabinose induction, we see most of the cells in Q3. The distribution of cells shifted to Q4 when arabinose was added to the medium. We also see that the cells remained in Q3 even after arabinose induction. The bimodal distribution indicate that some cells got induced while other cells were in repressed state. <br />
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<img style="width:80%; display: block;<br />
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margin-right: auto" src="https://static.igem.org/mediawiki/2014/0/02/FACS_hkust.png" /><br />
<h5 style="font-size: 13px">Figure 1. Forward scatter intensity (FSC) versus GFP graphs for samples with P<sub>BAD</sub> promoter regulating GFP generator. </h5><br />
<h6 style= "font-size: 13px"> All samples were inoculated in M9 minimal salt medium overnight in various arabinose concentrations (%w/v). The samples were diluted around 10 fold the next day. Sample were fixed and the fluorescence was measured using flow cytometer. The graphs were plotted for the control constructs, pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (-) and pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (<a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>) in the absence of arabinose. FSC versus GFP graphs for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>(<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>) in 0, 0.2 and 1.0% arabinose concentration were plotted. Each set of graphs were obtained for three different cell strains, DH10B, DH5α and BW25113. </h6> <br><br><br />
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<p>The distribution can also give us some idea of P<sub>BAD</sub> promoter leakage in different cells strain. If the data points are on or near the boundary between Q3 and Q4, we know that the promoter is leaky. The results indicate that the promoter is relatively more leaky in DH10B compared to other cell strains. <br><br><br />
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The percentages of cells in each Q3 and Q4 are highlighted in Figure 2. After addition of arabinose, we clearly see more percentage in Q4. Around 80% of the cells for DH10B and BW25113 are in Q4. Relatively lower percentage of cells in Q4 is observed for DH5α. Only around 60 to 70% of cells were in Q4 after arabinose induction. The shift in the distribution from Q3 to Q4 also corresponded to the increase of RPU across different arabinose concentration. At 0% arabinose concentration, for all three strain, higher percentage of cells are in Q3 and therefore the RPU is low. After arabinose induction, more cells are in Q4 and RPU is high. The percentage of cells in Q4 levels off for arabinose concentration that we have tested. The trend was observed for RPU measurement. <br><br><br />
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We can also conclude that even after maximum period of induction (overnight induction) of arabinose, we still see some cells uninduced (in Q3). <br><br><br />
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<img style="width:80%; display: block;<br />
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margin-right: auto" src="https://static.igem.org/mediawiki/2014/8/88/Percentageofcells_pbad_HKUST.png" /><br />
<h5 style="font-size: 13px">Figure 2. The percentage of cells in induced and uninduced state, and RPU across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Q3 and Q4 represent the 3<sup>rd</sup> and 4<sup>th</sup> quadrants of the forward scatter versus GFP curve mentioned in Figure 2. The experimental condition was same as the procedure mentioned in the caption of the Figure 1. The left y-axis is for the percent of cells in Q3 and Q4 while the right y-axis is for RPU. Graphs depict the triplicate mean ± standard deviation. (A) Graph for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in DH10B. (B) Graph for pSBK3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in DH5α. (C) Graph for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in BW25113. </h6> <br><br><br />
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<p><br />
<b><u>RPU of PBAD in different cell strains</u></b><br><br />
In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D graph while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another strain, BW25113, which is commonly used by synthetic biologists. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). <br><br><br />
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<img style="width:80%; display: block;<br />
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margin-right: auto" src="https://static.igem.org/mediawiki/2014/a/ab/Pbad_RPU_3strains.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using flow cytometer. The graph represent triplicate mean ±SD. </h6> <br><br><br />
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<p><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by other experimental factors such as genetic context. Because RPU is a ratio to fluorescence measurement, the effects caused by these factors can be minimized (See Methods for RPU calculation). <br><br><br />
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We observed clearly a different promoter response among the three cell strains. The all-or-none response was observed for the strains, but the levels of RPU were different. For DH10B, RPU leveled off around 0.45. For BW25113, RPU leveled off around 0.3. For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. <br><br><br />
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The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α. All in all, all three strains produced all-or-none response. <br><br><br />
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For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
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<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The RPU was around 0.16 for DH10B which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were below RPU of 0.1. The lowest leakage was observed for DH5α. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
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<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to produce a 3-D graph. We, however, had some changes from the genetic context that Groningen 2011 used. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840</a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator). We also used a low copy pSB3K3 plasmid as the backbone instead of the high copy pSB1C3. We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 4. A and B) <br><br><br />
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<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 4. Fluorescence and OD600 measurements of DH10B and DH5α induced in different arabinose concentrations. </h5><br />
<h6 style= "font-size: 13px"> Triplicate of DH10B and DH5α samples were inoculated in deep well 96 well plate overnight in M9 minimal salt medium. Arabinose was added to match the final working concentration from 0 to 1.0 % (w/v) with 0.2% increments. Fluorescence and OD600 was measured every two hours for ten hours. (A) Increase of OD600 measurement for DH10B strain in different arabinose concentration. The graph represents triplicate mean ± SD (B) Increase of OD600 measurement for DH5α strain in different arabinose concentration. (C) Fluorescence VS arabinose concentration VS Time 3-D graph for DH10B. Each point represent triplicate mean. (D) Fluorescence VS arabinose concentration VS Time 3-D graph for DH5α. Each point represent triplicate mean. </h6> <br><br><br />
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The OD600 value reflects cell concentration. OD600 for both strain in different arabinose concentration increased exponentially. Growth rate for cells without arabinose induction (0%) was greatest for both DH10B and DH5α. For other concentration of arabinose induction the growth rates were similar for both of the cell strains (Figure 4. A and B). The similar growth rate across the 10 hour period indicates that the cell concentration for samples in different arabinose concentration increased similarly and therefore the cell concentration at each point of measurement was similar across different arabinose concentration. We can therefore assume that differences in the cell concentrations have minimal effect on the different fluorescence levels at different arabinose concentration. <br><br><br />
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Unlike what Groningen 2011 observed in their experiment, (<a href="http://parts.igem.org/Part:BBa_I0500:Experience">Experience page.</a>) we observed all-or-none response for both of the cell strains. For DH10B and DH5α the fluorescence levelled off at 0.2% arabinose concentration. In the process of subtracting autofluorescence, we got some negative corrected fluorescence. These region correspond to the blue areas of the 3-D graphs. <br />
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<div class='content_1'><h3>Discussion</h3><br />
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<p><i>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter </i><br><br><br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Groningen 2011 team did not provide with OD600 versus time graphs. Therefore it is hard to tell well there was variation in the cell growth under the context of Groningen 2011’s experiment. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a>(<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) showed gradual response to increasing arabinose concentration. <br><br><br />
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<i>Different genetic context could be responsible for the different response of the P<sub>BAD</sub> promoter. </i><br><br><br />
<br />
Due to lack of human-power, unfortunately, we could not produce 3-D graphs for cells with pSB1C3 backbone as controls. Assuming that Groningen 2011 result was valid, we can speculate that difference of the P<sub>BAD</sub> promoter response could have resulted from the different backbone that we have used to characterize the promoter. In fact, Cambridge 2011, who also observed all-or-none response (<a href="http://parts.igem.org/Part:BBa_I0500:Experience">Experience page.</a>) used pSB3K3 as backbone for their constructs. Without proper controls, it is hard to conclude <br />
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<div class='content_1'><h3>Methods</h3><br />
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<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
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2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to DH10B, DH5alpha, and BW25113.<br><br><br />
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3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
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4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
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<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
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2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
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3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
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4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
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5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
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6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
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Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
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- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
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<b>OR</b><br><br><br />
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- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
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7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
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<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
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2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
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3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
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4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
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5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
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6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
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<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br />
<br />
Based on this equation, the GFP synthesis rate (dF/dt) for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the experiment construct. GFP synthesis rate (dF/dt) for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the GFP synthesis rate/ABS of experiment with the GFP synthesis rate/ABS of the reference promoter.<br />
<br />
<br />
<br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
Based on this equation, the amount of fluorescence [F] for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the experiment construct. Amount of fluorescence [F] for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the fluorescence*growth rate of experiment with the fluorescence*growth rate of the reference promoter.<br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
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<u><b>References</b></u><br><br></p><br />
<p><br />
<br />
Schleif R. <i>AraC protein, regulation of the L-arabinose operon in Escherichia coli, and the light switch mechanism of AraC action.</i> FEMS Microbiol Rev (2010) 1–18.<br><br><br />
<br />
Khelbnikov, A., Datsenko, K., Skaug, T., Wanner, B., & Keasling, J. (2001).<i> Homogeneous expression of the P(BAD) promoter in Escherichia coli by constitutive expression of the low-affinity high-capacity AraE transporter. </i> Microbiology, 147(12), 3241-3247.<br><br><br />
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J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-18T01:08:46Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
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<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
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<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
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<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
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<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
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<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD. The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can activate the P<sub>BAD</sub> promoter (Schleif, 2010).</p><br />
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<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<p> <br />
<br />
There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter responded to the arabinose induction. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge results show that it was an “on-or-off” response. We wanted to analyze these problems so that the part could be more reliable for other users. Cambridge cited a paper that mentioned that variation in response could have resulted from cell strain variation Khelbnikov, 2001)</p><br />
<br />
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<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
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<p><u>All-or-none response observed for individual cells</u>.<br>Flow cytometry can measure the fluorescence of individual cells. The individual measurement of cell fluorescence can be plotted in a forward scatter (FSC) intensity value versus fluorescence graph. An arbitrary vertical line divided the region of the graph with low fluorescence (Q3) and high fluorescence (Q4). <br><br><br />
<br />
For the negative control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, we see cells distributed in Q3 while absent in Q4. Converse was true for the positive control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (GFP generator regulated by <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>). We see most cells distributed in Q4. For our experimental sample, (pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) similar trend could observed for three strains. At no arabinose induction, we see most of the cells in Q3. The distribution of cells shifted to Q4 when arabinose was added to the medium. We also see that the cells remained in Q3 even after arabinose induction. The bimodal distribution indicate that some cells got induced while other cells were in repressed state. <br />
<br />
</p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/0/02/FACS_hkust.png" /><br />
<h5 style="font-size: 13px">Figure 1. Forward scatter intensity (FSC) versus GFP graphs for samples with P<sub>BAD</sub> promoter regulating GFP generator. </h5><br />
<h6 style= "font-size: 13px"> All samples were inoculated in M9 minimal salt medium overnight in various arabinose concentrations (%w/v). The samples were diluted around 10 fold the next day. Sample were fixed and the fluorescence was measured using flow cytometer. The graphs were plotted for the control constructs, pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (-) and pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (<a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>) in the absence of arabinose. FSC versus GFP graphs for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>(<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>) in 0, 0.2 and 1.0% arabinose concentration were plotted. Each set of graphs were obtained for three different cell strains, DH10B, DH5α and BW25113. </h6> <br><br><br />
<br />
<br />
<p>The distribution can also give us some idea of P<sub>BAD</sub> promoter leakage in different cells strain. If the data points are on or near the boundary between Q3 and Q4, we know that the promoter is leaky. The results indicate that the promoter is relatively more leaky in DH10B compared to other cell strains. <br><br><br />
<br />
The percentages of cells in each Q3 and Q4 are highlighted in Figure 2. After addition of arabinose, we clearly see more percentage in Q4. Around 80% of the cells for DH10B and BW25113 are in Q4. Relatively lower percentage of cells in Q4 is observed for DH5α. Only around 60 to 70% of cells were in Q4 after arabinose induction. The shift in the distribution from Q3 to Q4 also corresponded to the increase of RPU across different arabinose concentration. At 0% arabinose concentration, for all three strain, higher percentage of cells are in Q3 and therefore the RPU is low. After arabinose induction, more cells are in Q4 and RPU is high. The percentage of cells in Q4 levels off for arabinose concentration that we have tested. The trend was observed for RPU measurement. <br><br><br />
<br />
We can also conclude that even after maximum period of induction (overnight induction) of arabinose, we still see some cells uninduced (in Q3). <br><br><br />
<br />
</p><br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/8/88/Percentageofcells_pbad_HKUST.png" /><br />
<h5 style="font-size: 13px">Figure 2. The percentage of cells in induced and uninduced state, and RPU across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Q3 and Q4 represent the 3<sup>rd</sup> and 4<sup>th</sup> quadrants of the forward scatter versus GFP curve mentioned in Figure 2. The experimental condition was same as the procedure mentioned in the caption of the Figure 1. The left y-axis is for the percent of cells in Q3 and Q4 while the right y-axis is for RPU. Graphs depict the triplicate mean ± standard deviation. (A) Graph for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in DH10B. (B) Graph for pSBK3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in DH5α. (C) Graph for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in BW25113. </h6> <br><br><br />
<br />
<p><br />
<b><u>RPU of PBAD in different cell strains</u></b><br><br />
In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D graph while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another strain, BW25113, which is commonly used by synthetic biologists. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). <br><br><br />
</p><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/a/ab/Pbad_RPU_3strains.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using flow cytometer. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by other experimental factors such as genetic context. Because RPU is a ratio to fluorescence measurement, the effects caused by these factors can be minimized (See Methods for RPU calculation). <br><br><br />
<br />
We observed clearly a different promoter response among the three cell strains. The all-or-none response was observed for the strains, but the levels of RPU were different. For DH10B, RPU leveled off around 0.45. For BW25113, RPU leveled off around 0.3. For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The RPU was around 0.16 for DH10B which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were below RPU of 0.1. The lowest leakage was observed for DH5α. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to produce a 3-D graph. We, however, had some changes from the genetic context that Groningen 2011 used. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840</a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator). We also used a low copy pSB3K3 plasmid as the backbone instead of the high copy pSB1C3. We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 4. A and B) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 4. Fluorescence and OD600 measurements of DH10B and DH5α induced in different arabinose concentrations. </h5><br />
<h6 style= "font-size: 13px"> Triplicate of DH10B and DH5α samples were inoculated in deep well 96 well plate overnight in M9 minimal salt medium. Arabinose was added to match the final working concentration from 0 to 1.0 % (w/v) with 0.2% increments. Fluorescence and OD600 was measured every two hours for ten hours. (A) Increase of OD600 measurement for DH10B strain in different arabinose concentration. The graph represents triplicate mean ± SD (B) Increase of OD600 measurement for DH5α strain in different arabinose concentration. (C) Fluorescence VS arabinose concentration VS Time 3-D graph for DH10B. Each point represent triplicate mean. (D) Fluorescence VS arabinose concentration VS Time 3-D graph for DH5α. Each point represent triplicate mean. </h6> <br><br><br />
</p><br />
<br />
<p><br />
The OD600 value reflects cell concentration. OD600 for both strain in different arabinose concentration increased exponentially. Growth rate for cells without arabinose induction (0%) was greatest for both DH10B and DH5α. For other concentration of arabinose induction the growth rates were similar for both of the cell strains (Figure 4. A and B). The similar growth rate across the 10 hour period indicates that the cell concentration for samples in different arabinose concentration increased similarly and therefore the cell concentration at each point of measurement was similar across different arabinose concentration. We can therefore assume that differences in the cell concentrations have minimal effect on the different fluorescence levels at different arabinose concentration. <br><br><br />
<br />
Unlike what Groningen 2011 observed in their experiment, (<a href="http://parts.igem.org/Part:BBa_I0500:Experience">Experience page.</a>) we observed all-or-none response for both of the cell strains. For DH10B and DH5α the fluorescence levelled off at 0.2% arabinose concentration. In the process of subtracting autofluorescence, we got some negative corrected fluorescence. These region correspond to the blue areas of the 3-D graphs. <br />
<br />
<br />
</p><br />
<br />
<br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p><i>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter </i><br><br><br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Groningen 2011 team did not provide with OD600 versus time graphs. Therefore it is hard to tell well there was variation in the cell growth under the context of Groningen 2011’s experiment. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a>(<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) showed gradual response to increasing arabinose concentration. <br><br><br />
<br />
<i>Different genetic context could be responsible for the different response of the P<sub>BAD</sub> promoter. </i><br><br><br />
<br />
Due to lack of human-power, unfortunately, we could not produce 3-D graphs for cells with pSB1C3 backbone as controls. Assuming that Groningen 2011 result was valid, we can speculate that difference of the P<sub>BAD</sub> promoter response could have resulted from the different backbone that we have used to characterize the promoter. In fact, Cambridge 2011, who also observed all-or-none response (<a href="http://parts.igem.org/Part:BBa_I0500:Experience">Experience page.</a>) used pSB3K3 as backbone for their constructs. Without proper controls, it is hard to conclude <br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br />
<br />
Based on this equation, the GFP synthesis rate (dF/dt) for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the experiment construct. GFP synthesis rate (dF/dt) for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the GFP synthesis rate/ABS of experiment with the GFP synthesis rate/ABS of the reference promoter.<br />
<br />
<br />
<br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
Based on this equation, the amount of fluorescence [F] for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the experiment construct. Amount of fluorescence [F] for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the fluorescence*growth rate of experiment with the fluorescence*growth rate of the reference promoter.<br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-18T01:08:11Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
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<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
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</a></div><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
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</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
</tr><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
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<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD. The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can activate the P<sub>BAD</sub> promoter (Schleif, 2010).</p><br />
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<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
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<p> <br />
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There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter responded to the arabinose induction. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge results show that it was an “on-or-off” response. We wanted to analyze these problems so that the part could be more reliable for other users. Cambridge cited a paper that mentioned that variation in response could have resulted from cell strain variation Khelbnikov, 2001)</p><br />
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<div class='content_1'><h3>Results</h3><br />
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<p><u>All-or-none response observed for individual cells</u>.<br>Flow cytometry can measure the fluorescence of individual cells. The individual measurement of cell fluorescence can be plotted in a forward scatter (FSC) intensity value versus fluorescence graph. An arbitrary vertical line divided the region of the graph with low fluorescence (Q3) and high fluorescence (Q4). <br><br><br />
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For the negative control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, we see cells distributed in Q3 while absent in Q4. Converse was true for the positive control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (GFP generator regulated by <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>). We see most cells distributed in Q4. For our experimental sample, (pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) similar trend could observed for three strains. At no arabinose induction, we see most of the cells in Q3. The distribution of cells shifted to Q4 when arabinose was added to the medium. We also see that the cells remained in Q3 even after arabinose induction. The bimodal distribution indicate that some cells got induced while other cells were in repressed state. <br />
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<img style="width:80%; display: block;<br />
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<h5 style="font-size: 13px">Figure 1. Forward scatter intensity (FSC) versus GFP graphs for samples with P<sub>BAD</sub> promoter regulating GFP generator. </h5><br />
<h6 style= "font-size: 13px"> All samples were inoculated in M9 minimal salt medium overnight in various arabinose concentrations (%w/v). The samples were diluted around 10 fold the next day. Sample were fixed and the fluorescence was measured using flow cytometer. The graphs were plotted for the control constructs, pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (-) and pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (<a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>) in the absence of arabinose. FSC versus GFP graphs for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>(<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>) in 0, 0.2 and 1.0% arabinose concentration were plotted. Each set of graphs were obtained for three different cell strains, DH10B, DH5α and BW25113. </h6> <br><br><br />
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<p>The distribution can also give us some idea of P<sub>BAD</sub> promoter leakage in different cells strain. If the data points are on or near the boundary between Q3 and Q4, we know that the promoter is leaky. The results indicate that the promoter is relatively more leaky in DH10B compared to other cell strains. <br><br><br />
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The percentages of cells in each Q3 and Q4 are highlighted in Figure 2. After addition of arabinose, we clearly see more percentage in Q4. Around 80% of the cells for DH10B and BW25113 are in Q4. Relatively lower percentage of cells in Q4 is observed for DH5α. Only around 60 to 70% of cells were in Q4 after arabinose induction. The shift in the distribution from Q3 to Q4 also corresponded to the increase of RPU across different arabinose concentration. At 0% arabinose concentration, for all three strain, higher percentage of cells are in Q3 and therefore the RPU is low. After arabinose induction, more cells are in Q4 and RPU is high. The percentage of cells in Q4 levels off for arabinose concentration that we have tested. The trend was observed for RPU measurement. <br><br><br />
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We can also conclude that even after maximum period of induction (overnight induction) of arabinose, we still see some cells uninduced (in Q3). <br><br><br />
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<img style="width:80%; display: block;<br />
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<h5 style="font-size: 13px">Figure 2. The percentage of cells in induced and uninduced state, and RPU across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Q3 and Q4 represent the 3<sup>rd</sup> and 4<sup>th</sup> quadrants of the forward scatter versus GFP curve mentioned in Figure 2. The experimental condition was same as the procedure mentioned in the caption of the Figure 1. The left y-axis is for the percent of cells in Q3 and Q4 while the right y-axis is for RPU. Graphs depict the triplicate mean ± standard deviation. (A) Graph for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in DH10B. (B) Graph for pSBK3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in DH5α. (C) Graph for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in BW25113. </h6> <br><br><br />
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<u>RPU of PBAD in different cell strains</u><br><br />
In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D graph while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another strain, BW25113, which is commonly used by synthetic biologists. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). <br><br><br />
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<img style="width:80%; display: block;<br />
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<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using flow cytometer. The graph represent triplicate mean ±SD. </h6> <br><br><br />
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We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by other experimental factors such as genetic context. Because RPU is a ratio to fluorescence measurement, the effects caused by these factors can be minimized (See Methods for RPU calculation). <br><br><br />
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We observed clearly a different promoter response among the three cell strains. The all-or-none response was observed for the strains, but the levels of RPU were different. For DH10B, RPU leveled off around 0.45. For BW25113, RPU leveled off around 0.3. For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. <br><br><br />
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The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α. All in all, all three strains produced all-or-none response. <br><br><br />
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For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
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<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The RPU was around 0.16 for DH10B which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were below RPU of 0.1. The lowest leakage was observed for DH5α. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
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<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to produce a 3-D graph. We, however, had some changes from the genetic context that Groningen 2011 used. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840</a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator). We also used a low copy pSB3K3 plasmid as the backbone instead of the high copy pSB1C3. We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 4. A and B) <br><br><br />
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<img style="width:80%; display: block;<br />
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<h5 style="font-size: 13px">Figure 4. Fluorescence and OD600 measurements of DH10B and DH5α induced in different arabinose concentrations. </h5><br />
<h6 style= "font-size: 13px"> Triplicate of DH10B and DH5α samples were inoculated in deep well 96 well plate overnight in M9 minimal salt medium. Arabinose was added to match the final working concentration from 0 to 1.0 % (w/v) with 0.2% increments. Fluorescence and OD600 was measured every two hours for ten hours. (A) Increase of OD600 measurement for DH10B strain in different arabinose concentration. The graph represents triplicate mean ± SD (B) Increase of OD600 measurement for DH5α strain in different arabinose concentration. (C) Fluorescence VS arabinose concentration VS Time 3-D graph for DH10B. Each point represent triplicate mean. (D) Fluorescence VS arabinose concentration VS Time 3-D graph for DH5α. Each point represent triplicate mean. </h6> <br><br><br />
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The OD600 value reflects cell concentration. OD600 for both strain in different arabinose concentration increased exponentially. Growth rate for cells without arabinose induction (0%) was greatest for both DH10B and DH5α. For other concentration of arabinose induction the growth rates were similar for both of the cell strains (Figure 4. A and B). The similar growth rate across the 10 hour period indicates that the cell concentration for samples in different arabinose concentration increased similarly and therefore the cell concentration at each point of measurement was similar across different arabinose concentration. We can therefore assume that differences in the cell concentrations have minimal effect on the different fluorescence levels at different arabinose concentration. <br><br><br />
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Unlike what Groningen 2011 observed in their experiment, (<a href="http://parts.igem.org/Part:BBa_I0500:Experience">Experience page.</a>) we observed all-or-none response for both of the cell strains. For DH10B and DH5α the fluorescence levelled off at 0.2% arabinose concentration. In the process of subtracting autofluorescence, we got some negative corrected fluorescence. These region correspond to the blue areas of the 3-D graphs. <br />
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<div class='content_1'><h3>Discussion</h3><br />
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<p><i>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter </i><br><br><br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Groningen 2011 team did not provide with OD600 versus time graphs. Therefore it is hard to tell well there was variation in the cell growth under the context of Groningen 2011’s experiment. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a>(<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) showed gradual response to increasing arabinose concentration. <br><br><br />
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<i>Different genetic context could be responsible for the different response of the P<sub>BAD</sub> promoter. </i><br><br><br />
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Due to lack of human-power, unfortunately, we could not produce 3-D graphs for cells with pSB1C3 backbone as controls. Assuming that Groningen 2011 result was valid, we can speculate that difference of the P<sub>BAD</sub> promoter response could have resulted from the different backbone that we have used to characterize the promoter. In fact, Cambridge 2011, who also observed all-or-none response (<a href="http://parts.igem.org/Part:BBa_I0500:Experience">Experience page.</a>) used pSB3K3 as backbone for their constructs. Without proper controls, it is hard to conclude <br />
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<div class='content_1'><h3>Methods</h3><br />
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<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
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2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to DH10B, DH5alpha, and BW25113.<br><br><br />
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3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
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4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
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<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
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2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
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3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
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4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
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5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
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6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
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Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
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- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
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<b>OR</b><br><br><br />
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- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
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7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
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<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
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2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
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3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
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4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
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5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
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6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
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<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
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Based on this equation, the GFP synthesis rate (dF/dt) for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the experiment construct. GFP synthesis rate (dF/dt) for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the reference promoter construct.<br><br><br />
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Then, RPU is obtained by dividing the GFP synthesis rate/ABS of experiment with the GFP synthesis rate/ABS of the reference promoter.<br />
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<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
Based on this equation, the amount of fluorescence [F] for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the experiment construct. Amount of fluorescence [F] for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the fluorescence*growth rate of experiment with the fluorescence*growth rate of the reference promoter.<br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-18T00:52:51Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
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<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
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</td><br />
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<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
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<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
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<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD. The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can activate the P<sub>BAD</sub> promoter (Schleif, 2010).</p><br />
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<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
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<p> <br />
<br />
There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter responded to the arabinose induction. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge results show that it was an “on-or-off” response. We wanted to analyze these problems so that the part could be more reliable for other users. Cambridge cited a paper that mentioned that variation in response could have resulted from cell strain variation Khelbnikov, 2001)</p><br />
<br />
<br><br />
</div><br />
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</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p><u>All-or-none response observed for individual cells</u>. Flow cytometry can measure the fluorescence of individual cells. The individual measurement of cell fluorescence can be plotted in a forward scatter (FSC) intensity value versus fluorescence graph. An arbitrary vertical line divided the region of the graph with low fluorescence (Q3) and high fluorescence (Q4). <br><br><br />
<br />
For the negative control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, we see cells distributed in Q3 while absent in Q4. Converse was true for the positive control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (GFP generator regulated by <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>). We see most cells distributed in Q4. For our experimental sample, (pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) similar trend could observed for three strains. At no arabinose induction, we see most of the cells in Q3. The distribution of cells shifted to Q4 when arabinose was added to the medium. We also see that the cells remained in Q3 even after arabinose induction. The bimodal distribution indicate that some cells got induced while other cells were in repressed state. <br />
<br />
</p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/0/02/FACS_hkust.png" /><br />
<h5 style="font-size: 13px">Figure 1. Forward scatter intensity (FSC) versus GFP graphs for samples with P<sub>BAD</sub> promoter regulating GFP generator. </h5><br />
<h6 style= "font-size: 13px"> All samples were inoculated in M9 minimal salt medium overnight in various arabinose concentrations (%w/v). The samples were diluted around 10 fold the next day. Sample were fixed and the fluorescence was measured using flow cytometer. The graphs were plotted for the control constructs, pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (-) and pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (<a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>) in the absence of arabinose. FSC versus GFP graphs for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>(<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>) in 0, 0.2 and 1.0% arabinose concentration were plotted. Each set of graphs were obtained for three different cell strains, DH10B, DH5α and BW25113. </h6> <br><br><br />
<br />
<br />
<p>The distribution can also give us some idea of P<sub>BAD</sub> promoter leakage in different cells strain. If the data points are on or near the boundary between Q3 and Q4, we know that the promoter is leaky. The results indicate that the promoter is relatively more leaky in DH10B compared to other cell strains. <br><br><br />
<br />
The percentages of cells in each Q3 and Q4 are highlighted in Figure 2. After addition of arabinose, we clearly see more percentage in Q4. Around 80% of the cells for DH10B and BW25113 are in Q4. Relatively lower percentage of cells in Q4 is observed for DH5α. Only around 60 to 70% of cells were in Q4 after arabinose induction. The shift in the distribution from Q3 to Q4 also corresponded to the increase of RPU across different arabinose concentration. At 0% arabinose concentration, for all three strain, higher percentage of cells are in Q3 and therefore the RPU is low. After arabinose induction, more cells are in Q4 and RPU is high. The percentage of cells in Q4 levels off for arabinose concentration that we have tested. The trend was observed for RPU measurement. <br><br><br />
<br />
We can also conclude that even after maximum period of induction (overnight induction) of arabinose, we still see some cells uninduced (in Q3). <br><br><br />
<br />
</p><br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/8/88/Percentageofcells_pbad_HKUST.png" /><br />
<h5 style="font-size: 13px">Figure 2. The percentage of cells in induced and uninduced state, and RPU across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Q3 and Q4 represent the 3<sup>rd</sup> and 4<sup>th</sup> quadrants of the forward scatter versus GFP curve mentioned in Figure 2. The experimental condition was same as the procedure mentioned in the caption of the Figure 1. The left y-axis is for the percent of cells in Q3 and Q4 while the right y-axis is for RPU. Graphs depict the triplicate mean ± standard deviation. (A) Graph for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in DH10B. (B) Graph for pSBK3K3-<a href="http://parts.igem.org/Part:BBa_I0500">-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in DH5α. (C) Graph for pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> in BW25113. </h6> <br><br><br />
<br />
<p><br />
<u>RPU of PBAD in different cell strains</u><br><br />
In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D graph while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another strain, BW25113, which is commonly used by synthetic biologists. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). <br><br><br />
</p><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/a/ab/Pbad_RPU_3strains.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using flow cytometer. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
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<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br />
<br />
Based on this equation, the GFP synthesis rate (dF/dt) for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the experiment construct. GFP synthesis rate (dF/dt) for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the GFP synthesis rate/ABS of experiment with the GFP synthesis rate/ABS of the reference promoter.<br />
<br />
<br />
<br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
Based on this equation, the amount of fluorescence [F] for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the experiment construct. Amount of fluorescence [F] for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the fluorescence*growth rate of experiment with the fluorescence*growth rate of the reference promoter.<br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
<br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/File:Pbad_RPU_3strains.pngFile:Pbad RPU 3strains.png2014-10-18T00:51:43Z<p>Jordyevan: </p>
<hr />
<div></div>Jordyevanhttp://2014.igem.org/File:Percentageofcells_pbad_HKUST.pngFile:Percentageofcells pbad HKUST.png2014-10-18T00:46:56Z<p>Jordyevan: </p>
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<div></div>Jordyevanhttp://2014.igem.org/File:FACS_hkust.pngFile:FACS hkust.png2014-10-18T00:41:03Z<p>Jordyevan: </p>
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<div></div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-18T00:39:04Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
<html><head><br />
<br />
</head></html><br />
|<br />
<html><body><br />
<div id="content_container"><br />
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<div id="description_area"><br />
<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<!-- end of one row of content , two column--><br />
<br />
</div><br />
</div><br />
<br><br><br />
<br />
<br />
<div id="content_container"><br />
<!-- one row of content , two column one picture right--><br />
<div id="description_area"><br />
<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD. The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can activate the P<sub>BAD</sub> promoter (Schleif, 2010).</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p> <br />
<br />
There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter responded to the arabinose induction. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge results show that it was an “on-or-off” response. We wanted to analyze these problems so that the part could be more reliable for other users. Cambridge cited a paper that mentioned that variation in response could have resulted from cell strain variation Khelbnikov, 2001)</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p><u>All-or-none response observed for individual cells</u>. Flow cytometry can measure the fluorescence of individual cells. The individual measurement of cell fluorescence can be plotted in a forward scatter (FSC) intensity value versus fluorescence graph. An arbitrary vertical line divided the region of the graph with low fluorescence (Q3) and high fluorescence (Q4). <br><br><br />
<br />
For the negative control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, we see cells distributed in Q3 while absent in Q4. Converse was true for the positive control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (GFP generator regulated by <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>). We see most cells distributed in Q4. For our experimental sample, (pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) similar trend could observed for three strains. At no arabinose induction, we see most of the cells in Q3. The distribution of cells shifted to Q4 when arabinose was added to the medium. We also see that the cells remained in Q3 even after arabinose induction. The bimodal distribution indicate that some cells got induced while other cells were in repressed state. <br />
<br />
</p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br />
<br />
Based on this equation, the GFP synthesis rate (dF/dt) for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the experiment construct. GFP synthesis rate (dF/dt) for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the GFP synthesis rate/ABS of experiment with the GFP synthesis rate/ABS of the reference promoter.<br />
<br />
<br />
<br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
Based on this equation, the amount of fluorescence [F] for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the experiment construct. Amount of fluorescence [F] for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the fluorescence*growth rate of experiment with the fluorescence*growth rate of the reference promoter.<br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-18T00:37:12Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
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</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD. The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate pathway. The Pc promoter which is adjacent to the <p>P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity of the <p>P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can activate the <p>P<sub>BAD</sub> promoter (Schleif, 2010).</p><br />
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<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
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<p> <br />
<br />
There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter responded to the arabinose induction. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge results show that it was an “on-or-off” response. We wanted to analyze these problems so that the part could be more reliable for other users. Cambridge cited a paper that mentioned that variation in response could have resulted from cell strain variation Khelbnikov, 2001)</p><br />
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<div class='content_1'><h3>Results</h3><br />
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<p><u>All-or-none response observed for individual cells</u>. Flow cytometry can measure the fluorescence of individual cells. The individual measurement of cell fluorescence can be plotted in a forward scatter (FSC) intensity value versus fluorescence graph. An arbitrary vertical line divided the region of the graph with low fluorescence (Q3) and high fluorescence (Q4). <br><br><br />
<br />
For the negative control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, we see cells distributed in Q3 while absent in Q4. Converse was true for the positive control, DH10B transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (GFP generator regulated by <a href="http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>). We see most cells distributed in Q4. For our experimental sample, (pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) similar trend could observed for three strains. At no arabinose induction, we see most of the cells in Q3. The distribution of cells shifted to Q4 when arabinose was added to the medium. We also see that the cells remained in Q3 even after arabinose induction. The bimodal distribution indicate that some cells got induced while other cells were in repressed state. <br />
<br />
</p><br />
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<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
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<div class='content_1'><h3>Discussion</h3><br />
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<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
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<br />
<div class='content_1'><h3>Methods</h3><br />
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<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br />
<br />
Based on this equation, the GFP synthesis rate (dF/dt) for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the experiment construct. GFP synthesis rate (dF/dt) for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the GFP synthesis rate/ABS of experiment with the GFP synthesis rate/ABS of the reference promoter.<br />
<br />
<br />
<br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
Based on this equation, the amount of fluorescence [F] for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the experiment construct. Amount of fluorescence [F] for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the fluorescence*growth rate of experiment with the fluorescence*growth rate of the reference promoter.<br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T23:09:23Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
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<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
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<div class='content_1'><h3>Riboregulator Results</h3><br />
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<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
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<br />
<div class='content_1'><h3>Introduction</h3><br />
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<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
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<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
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<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
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<div class='content_1'><h3>Results</h3><br />
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<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
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<div class='content_1'><h3>Discussion</h3><br />
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<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
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<br />
<div class='content_1'><h3>Methods</h3><br />
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<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br />
<br />
Based on this equation, the GFP synthesis rate (dF/dt) for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the experiment construct. GFP synthesis rate (dF/dt) for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the GFP synthesis rate/ABS of experiment with the GFP synthesis rate/ABS of the reference promoter.<br />
<br />
<br />
<br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
Based on this equation, the amount of fluorescence [F] for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the experiment construct. Amount of fluorescence [F] for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the fluorescence*growth rate of experiment with the fluorescence*growth rate of the reference promoter.<br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T23:07:58Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
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<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
<br />
Based on this equation, the GFP synthesis rate (dF/dt) for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the experiment construct. GFP synthesis rate (dF/dt) for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the GFP synthesis rate/ABS of experiment with the GFP synthesis rate/ABS of the reference promoter.<br />
<br />
<br />
<br><br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
Based on this equation, the amount of fluorescence [F] for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the experiment construct. Amount of fluorescence [F] for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the fluorescence*growth rate of experiment with the fluorescence*growth rate of the reference promoter.<br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T23:05:55Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
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<br><br><br />
<br />
<br />
<div id="content_container"><br />
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<div id="description_area"><br />
<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
<br />
- Based on this equation, the GFP synthesis rate (dF/dt) for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the experiment construct. <br><br><br />
<br />
GFP synthesis rate (dF/dt) for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) divided by the ABS (which means the absorbance or OD600) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the GFP synthesis rate/ABS of experiment with the GFP synthesis rate/ABS of the reference promoter.<br />
<br />
<br />
<br><br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
- Based on this equation, the amount of fluorescence [F] for the experiment (<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the experiment construct. <br><br><br />
<br />
amount of fluorescence [F] for the reference promoter (<a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>) multiplied by the growth rate (μ) of the cells containing the reference promoter construct.<br><br><br />
<br />
Then, RPU is obtained by dividing the fluorescence*growth rate of experiment with the fluorescence*growth rate of the reference promoter.<br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:54:46Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
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<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
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</a></div><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
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</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
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</table><br />
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<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
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</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
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</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:46:15Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
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<br />
<br />
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<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>BAD</sub> and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>BAD</sub> and absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>BAD</sub> in different concentration of arabinose is shown. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>BAD</sub> promoter in the absence of arabinose (0%).<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>)<br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:42:10Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
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<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
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<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
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</td><br />
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</table><br />
</div><br />
<br />
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<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
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</td><br />
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</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
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<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a><br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
<br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:41:45Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
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<br />
<div id="content_container"><br />
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<div id="description_area"><br />
<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a><br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:40:59Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
<html><head><br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
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<br />
<br />
<div id="content_container"><br />
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<div id="description_area"><br />
<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
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</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br />
<br />
2. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I0500">BBa_I0500</a>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br> to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
3. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br />
4. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit to DH10B, DH5alpha, and BW25113.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium and arabinose with specific concentration (0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%) in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used <b>Envision Multilabel Reader</b>) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
<b>OR</b><br><br><br />
<br />
- When cells were in the mid-log phase, cells were fixed and fluorescence was measured using <b>FACS</b>. <br />
<br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing for data from Envision Multilabel Reader</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
</p><br />
<br />
<p><br><u><b>Data Processing for data from FACS</b></u><br><br><br />
1. RPU was calculated by first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a><br><br><br />
2. Dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
Equation is shown below:<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
3. For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterizationTeam:Hong Kong HKUST/pneumosensor/characterization2014-10-17T22:28:20Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<h2>Pneumosensor Characterization</h2><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379004"> σ<sup>x</sup>(BBa_K1379004) </a></h3><br />
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<p><br><b><u>Introduction</u></b> <br><br><br />
<p class="first_letter_enhanced">To test the functionality of &sigma;<sup>X</sup>, we first enable constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator, <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. <br />
The generator was then assembled with the standard promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, with either promoter P<sub>celA</sub> (<br />
Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <br />
<a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a>) and P<sub>comFA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>, <br />
w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a>). <i>E. coli</i> colonies holding the resulting<br />
constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101"><br />
BBa_J23101</a>, which is <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240<br />
</a> was used as the general negative control for background fluorescence. Measurement kits for P<sub>celA</sub> and P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used <br />
as negative controls for function of &sigma;<sup>X</sup>.<br />
<br><br />
</p><br />
<p><br><b><u>Results</u></b> <br><br></p><br />
<div class="content_image"><br />
<img src= "https://static.igem.org/mediawiki/2014/7/7f/PcelA%26comFA_macroscope.png" ><br />
<h5 style="font-size: 13px">Figure 1. P<sub>celA</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.</h5><br />
<h6 style= "font-size: 13px"> Only in the presence of &sigma;<sup>X</sup> would P<sub>celA</sub> and P<sub>comFA</sub> be turned on, as GFP expression could <br />
be seen when &sigma;<sup>X</sup> is present. Therefore, &sigma;<sup>X</sup> is functional. P<sub>celA</sub> and P<sub>comFA</sub> gave little GFP signal in the <br />
absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter BBa_J23101 in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.</h6><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379000">P<sub>celA</sub> (BBa_K1379000)</a> and <a href= "http://parts.igem.org/Part:BBa_K1379001">P<sub>comFA</sub> (BBa_K1379001) </a></h3><br />
<table class="content_table" align= "center" ><br />
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<br />
<p class="first_letter_enhanced"> <br><br>For characterization, P<sub>celA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>celA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a>. <br><br><br />
<br />
P<sub>comFA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>comFA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a>. <br><br><br />
<br />
Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>celA</sub> and P<sub>comFA</sub> Measurement Kits with and without the &sigma;<sup>X</sup> generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the negative control for background fluorescence. <br><br><br />
<br />
Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). <i>E. coli</i> DH10B strains holding the constructs with or without &sigma;<sup>X</sup> generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> measurement device <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was measured alongside. The measurement was done with 3 replicas. <br />
</div><br><br><br />
<br />
<div class= "content_area_one_row"><br />
<div class="content_image"><br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src= "https://static.igem.org/mediawiki/2014/8/8c/Wiki_hkust_pcelA_pcomFA.png"/><br><br />
<h5 style="font-size: 13px">Figure 2. P<sub>celA</sub> has 0.53 RPU and P<sub>comFA</sub> hsa 1.21 RPU when paired with &sigma;<sup>X</sup> generator.</h5><br />
<h6 style= "font-size: 13px"> P<sub>celA</sub> and P<sub>comFA</sub> was measured in reference to <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> constitutive promoter with and without &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. RPU shown was calculated from 3 replicas.</h6> <br><br><br />
<br />
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<div class='content_1'><h3>Characterization Method</h3><br />
<table class="content_table" align= "center" ><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"> <br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-σ<sup>x</sup> Generator-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br />
<br />
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a> to pSB3k3.<br><br><br />
<br />
2. Construct pSB3K3-σ<sup>x</sup> Generator-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> <br><br />
<br />
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a> to pSB3K3.<br><br><br />
<br />
3. Transforming pSB3K3-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
<br />
4. Transforming pSB3K3-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
5. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit<br><br><br />
<br />
6. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on the construction part. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used Envision Multilabel Reader) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
</p><br />
</div><br />
</td></tr></table></div><br />
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<td class= "content_cell" colspan= "2"><br />
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<p><br />
<br><br />
<u><b>References</b></u><br><br></p><br />
<p><br />
<br />
BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 and http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 <br />
<br><br><br />
<br />
Luo P., & Morrison D. (2003).<i> Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae</i>. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003<br />
<br><br><br />
<br />
Piotrowski A., Luo P., & Morrison D. (2009). <i>Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.</i> Journal of Bacteriology. doi:10.1128/JB.01750-08<br />
<br><br><br />
<br />
Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). <i>Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.</i> Molecular Systhetic Biology .doi:10.1038/msb.2013.58<br />
<br><br><br />
<br />
J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4<br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterizationTeam:Hong Kong HKUST/pneumosensor/characterization2014-10-17T22:25:57Z<p>Jordyevan: </p>
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<h2>Pneumosensor Characterization</h2><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379004"> σ<sup>x</sup>(BBa_K1379004) </a></h3><br />
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<p><br><b><u>Introduction</u></b> <br><br><br />
<p class="first_letter_enhanced">To test the functionality of &sigma;<sup>X</sup>, we first enable constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator, <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. <br />
The generator was then assembled with the standard promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, with either promoter P<sub>celA</sub> (<br />
Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <br />
<a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a>) and P<sub>comFA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>, <br />
w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a>). <i>E. coli</i> colonies holding the resulting<br />
constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101"><br />
BBa_J23101</a>, which is <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240<br />
</a> was used as the general negative control for background fluorescence. Measurement kits for P<sub>celA</sub> and P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used <br />
as negative controls for function of &sigma;<sup>X</sup>.<br />
<br><br />
</p><br />
<p><br><b><u>Results</u></b> <br><br></p><br />
<div class="content_image"><br />
<img src= "https://static.igem.org/mediawiki/2014/7/7f/PcelA%26comFA_macroscope.png" ><br />
<h5 style="font-size: 13px">Figure 1. P<sub>celA</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.</h5><br />
<h6 style= "font-size: 13px"> Only in the presence of &sigma;<sup>X</sup> would P<sub>celA</sub> and P<sub>comFA</sub> be turned on, as GFP expression could <br />
be seen when &sigma;<sup>X</sup> is present. Therefore, &sigma;<sup>X</sup> is functional. P<sub>celA</sub> and P<sub>comFA</sub> gave little GFP signal in the <br />
absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter BBa_J23101 in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.</h6><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379000">P<sub>celA</sub> (BBa_K1379000)</a> and <a href= "http://parts.igem.org/Part:BBa_K1379001">P<sub>comFA</sub> (BBa_K1379001) </a></h3><br />
<table class="content_table" align= "center" ><br />
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<div class= "content_area_two_row"><br />
<br />
<br />
<p class="first_letter_enhanced"> <br><br>For characterization, P<sub>celA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>celA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a>. <br><br><br />
<br />
P<sub>comFA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>comFA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a>. <br><br><br />
<br />
Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>celA</sub> and P<sub>comFA</sub> Measurement Kits with and without the &sigma;<sup>X</sup> generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the negative control for background fluorescence. <br><br><br />
<br />
Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). <i>E. coli</i> DH10B strains holding the constructs with or without &sigma;<sup>X</sup> generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> measurement device <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was measured alongside. The measurement was done with 3 replicas. <br />
</div><br><br><br />
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<div class= "content_area_one_row"><br />
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<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src= "https://static.igem.org/mediawiki/2014/8/8c/Wiki_hkust_pcelA_pcomFA.png"/><br><br />
<h5 style="font-size: 13px">Figure 2. P<sub>celA</sub> has 0.53 RPU and P<sub>comFA</sub> hsa 1.21 RPU when paired with &sigma;<sup>X</sup> generator.</h5><br />
<h6 style= "font-size: 13px"> P<sub>celA</sub> and P<sub>comFA</sub> was measured in reference to <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> constitutive promoter with and without &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. RPU shown was calculated from 3 replicas.</h6> <br><br><br />
<br />
</div><br />
<br />
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<div class='content_1'><h3>Characterization Method</h3><br />
<table class="content_table" align= "center" ><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"> <br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct pSB3K3-σ<sup>x</sup> Generator-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br />
<br />
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a> to pSB3k3.<br><br><br />
<br />
2. Construct pSB3K3-σ<sup>x</sup> Generator-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> <br><br />
<br />
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a> to pSB3K3.<br><br><br />
<br />
3. Transforming pSB3K3-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
<br />
4. Transforming pSB3K3-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
5. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit<br><br><br />
<br />
6. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on procedure number 1. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used Envision Multilabel Reader) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
</p><br />
</div><br />
</td></tr></table></div><br />
<br><br><br />
<br />
<br />
<br />
<!-- end of one row of content , two column one picture left--><br />
<br />
<div class='content_1'><br />
<table class="content_table" align= "center" ><br />
<tr class= "content_row" ><br />
<td class= "content_cell" colspan= "2"><br />
<div class= "content_area_two_row"><br />
<p><br />
<br><br />
<u><b>References</b></u><br><br></p><br />
<p><br />
<br />
BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 and http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 <br />
<br><br><br />
<br />
Luo P., & Morrison D. (2003).<i> Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae</i>. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003<br />
<br><br><br />
<br />
Piotrowski A., Luo P., & Morrison D. (2009). <i>Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.</i> Journal of Bacteriology. doi:10.1128/JB.01750-08<br />
<br><br><br />
<br />
Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). <i>Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.</i> Molecular Systhetic Biology .doi:10.1038/msb.2013.58<br />
<br><br><br />
<br />
J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4<br />
<br />
<br />
<br />
<br />
<br />
<br />
</p> <br />
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</tr><br />
</table><br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:15:19Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
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<div class='content_1'><h3>Introduction</h3><br />
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<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
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</a></div><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
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</table><br />
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<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
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</table><br />
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<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>, pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>, and pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>, and dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
</p><br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:13:32Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
<html><head><br />
<br />
</head></html><br />
|<br />
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<div id="content_container"><br />
<br />
<div id="description_area"><br />
<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
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</table><br />
</div><br />
<br />
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<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>, pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0260">BBa_I0260 </a>, and pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>, and dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
</p><br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:12:30Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
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<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
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<br><br />
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<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part:BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
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<div class='content_1'><h3>Results</h3><br />
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<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
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<div class='content_1'><h3>Discussion</h3><br />
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<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
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<div class='content_1'><h3>Methods</h3><br />
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<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>, pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0260">BBa_I0260 </a>, and pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a>, and dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a>.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part:BBa_E0240">BBa_E0240 </a> and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part:BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:07:37Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
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<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
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</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part:BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
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<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part: BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part: BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part: >BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part: BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>, pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0260">BBa_I0260 </a>, and pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>, and dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I20260">BBa_I20260 </a>.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-BBa_E0240 and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:07:05Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
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<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
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<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part: BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part: BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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</table><br />
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<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part: BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part: >BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part: BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>, pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0260">BBa_I0260 </a>, and pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>, and dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I20260">BBa_I20260 </a>.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-BBa_E0240 and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:06:31Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
<html><head><br />
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</head></html><br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part:BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part:BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part: BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part:BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part:BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part: BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
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<br />
<br />
<div id="content_container"><br />
<!-- one row of content , two column one picture right--><br />
<div id="description_area"><br />
<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part: BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part: >BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part: BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>, pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0260">BBa_I0260 </a>, and pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>, and dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I20260">BBa_I20260 </a>.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-BBa_E0240 and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:05:35Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
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<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
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<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part: BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part: BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part: BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part: BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part:BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part: BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<div id="description_area"><br />
<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
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</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part: BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part: >BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part: BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>, pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0260">BBa_I0260 </a>, and pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>, and dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I20260">BBa_I20260 </a>.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-BBa_E0240 and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T22:02:42Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
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</a></div><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part: BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 <a href="http://parts.igem.org/Part: BBa_J01080">BBa_J01080 </a> Key3 <a href="http://parts.igem.org/Part: BBa_J01086">BBa_J01086 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock <a href="http://parts.igem.org/Part: BBa_K175031">BBa_K175031</a> Key for medium lock <a href="http://parts.igem.org/Part: BBa_K175032">BBa_K175032 </a> </td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part: BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008 </a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part: BBa_J01010">BBa_J01010 </a> Key 1<a href="http://parts.igem.org/Part: BBa_J01008">BBa_J01008</a></td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 <a href="http://parts.igem.org/Part: BBa_J01010">BBa_J01010 </a> Key 1 <a href="http://parts.igem.org/Part: BBa_J010080">BBa_J01008 </a> </td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter <a href="http://parts.igem.org/Part: BBa_J23102">BBa_J23102 </a> to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> <br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<div id="description_area"><br />
<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
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</td><br />
</tr><br />
</table><br />
</div><br />
<br />
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<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was <a href="http://parts.igem.org/Part: BBa_B0015">BBa_B0015 </a> because of two reasons. First, <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a>, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a> needed debugging. <a href="http://parts.igem.org/Part: BBa_I0500">BBa_I0500 </a>, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using <a href="http://parts.igem.org/Part: >BBa_E0840">BBa_E0840 </a> (GFP generator), we used <a href="http://parts.igem.org/Part: BBa_E0240">BBa_E0240 </a> (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0840">BBa_E0840 </a>) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>, pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0260">BBa_I0260 </a>, and pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a>, and dividing the fluorescence of cells containing pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-<a href="http://parts.igem.org/Part: >BBa_E0240">BBa_E0240 </a> with cells containing pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I20260">BBa_I20260 </a>.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I0500">BBa_I0500 </a>-BBa_E0240 and cells transformed with pSB3K3-<a href="http://parts.igem.org/Part: >BBa_I20260">BBa_I20260 </a> had same similar growth rate. <br />
<br />
</p><br />
<br />
<br><br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T20:41:00Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
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</a></div><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 (BBa_J01080) Key3 (BBa_J01086)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock (BBa_K175031)Key for medium lock (BBa_K175032)</td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter (BBa_J23102) to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter (BBa_I0500)<br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
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</table><br />
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<div id="content_container"><br />
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<div id="description_area"><br />
<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter BBa_I0500 in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was BBa_I0500 because of two reasons. First, BBa_I0500, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, BBa_I0500 needed debugging. BBa_I0500, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using BBa_E0840 (GFP generator), we used BBa_E0240 (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
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</td><br />
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</table><br />
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<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing <a href= "http://parts.igem.org/Part:BBa_K607036">BBa_K607036</a> (BBa_I0500-BBaE0840) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-BBa_E0240, pSB3K3-BBa_I20260, and pSB3K3-BBa_I0500-BBa_E0240. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-BBa_E0240, and dividing the fluorescence of cells containing pSB3K3-BBa_I0500-BBa_E0240 with cells containing pSB3K3-BBa_I20260.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-BBa_I0500-BBa_E0240 and cells transformed with pSB3K3-BBa_I20260 had same similar growth rate. <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T20:39:23Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 (BBa_J01080) Key3 (BBa_J01086)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock (BBa_K175031)Key for medium lock (BBa_K175032)</td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter (BBa_J23102) to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible P<sub>BAD</sub> promoter (BBa_I0500)<br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
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<div id="description_area"><br />
<br><h2>P<sub>BAD</sub> Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>P<sub>BAD</sub> promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the P<sub>BAD</sub> promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the P<sub>BAD</sub> promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the P<sub>BAD</sub> promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>P<sub>BAD</sub> promoter BBa_I0500 in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several P<sub>BAD</sub> promoters in the Part Registry. The promoter that we were interested was BBa_I0500 because of two reasons. First, BBa_I0500, along with P<sub>BAD</sub>, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of P<sub>BAD</sub> can only work on strain that are AraC+. By coupling the araC gene with the P<sub>BAD</sub> promoter, we can be free from such restraints. Second, BBa_I0500 needed debugging. BBa_I0500, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the P<sub>BAD</sub> promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of P<sub>BAD</sub> promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of P<sub>BAD</sub> in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of P<sub>BAD</sub> in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for P<sub>BAD</sub> promoter in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of P<sub>BAD</sub> promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the P<sub>BAD</sub> promoter, instead of using BBa_E0840 (GFP generator), we used BBa_E0240 (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
</p><br />
<br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Discussion</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Groningen 2011 results may not truly represent the gradual induction of P<sub>BAD</sub> promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing BBa_K607036 (BBa_I0500-BBaE0840) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<div class='content_1'><h3>Methods</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-BBa_E0240, pSB3K3-BBa_I20260, and pSB3K3-BBa_I0500-BBa_E0240. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-BBa_E0240, and dividing the fluorescence of cells containing pSB3K3-BBa_I0500-BBa_E0240 with cells containing pSB3K3-BBa_I20260.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-BBa_I0500-BBa_E0240 and cells transformed with pSB3K3-BBa_I20260 had same similar growth rate. <br />
<br />
</p><br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T20:33:31Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
<html><head><br />
<br />
</head></html><br />
|<br />
<html><body><br />
<div id="content_container"><br />
<br />
<div id="description_area"><br />
<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 (BBa_J01080) Key3 (BBa_J01086)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock (BBa_K175031)Key for medium lock (BBa_K175032)</td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter (BBa_J23102) to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible PBAD promoter (BBa_I0500)<br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
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<div id="description_area"><br />
<br><h2>PBAD Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>PBAD promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the PBAD promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the PBAD promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the PBAD promoter.</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<br />
<div class='content_1'><h3>PBAD promoter BBa_I0500 in Part Registry</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>There are several PBAD promoters in the Part Registry. The promoter that we were interested was BBa_I0500 because of two reasons. First, BBa_I0500, along with PBAD, has araC gene regulated by the Pc promoter. Without the AraC, the repression and induction of PBAD can only work on strain that are AraC+. By coupling the araC gene with the PBAD promoter, we can be free from such restraints. Second, BBa_I0500 needed debugging. BBa_I0500, although it is useful, it is not requestable because of inconsistency in sequencing. Also, in the experience page, two teams, Groningen 2011 and Cambridge 2011 had some discrepancy between how the promoter was induced. In brief, Groningen results show that the induction of the promoter by arabinose was gradual while Cambridge reported that it was more of an “on-or-off” induction. We wanted to solve these problems so that the part could be more reliable for other users</p><br />
<br />
<br><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<br />
<br />
<br />
<div class='content_1'><h3>Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to solve the discrepancy between Groningen 2011 and Cambridge 2011, we have calculated the Relative Promoter Unit of the PBAD promoter in three different cell strains across increasing arabinose concentration. The three strains that have chosen was: DH10B, BW25113, and DH5alpha. Groningen 2011 used cell strain DH5alpha to obtain the 3-D plot while Cambridge 2011 used BW27783. Unfortunately, we did not had access to the strain, so we had to use another similar strain BW25113. We thought that we had the capacity to test out another strain, so we added DH10B to the experiment. Only DH5alpha has araC in its genome. All other strains have deletion of araC (and other genes in the L-arabinose operon). </p><br />
<br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/6/68/Pbad_RPU_1_copy.png" /><br />
<h5 style="font-size: 13px">Figure 3. RPU of PBAD promoter in three different cell strains across different arabinose concentration. </h5><br />
<h6 style= "font-size: 13px"> Relative Promoter Unit of PBAD¬ promoter was calculated in three strains: DH10B, BW25113 and DH5alpha. Gradient arabinose concentration (% w/v) from 0% to 1.0% with 0.2% increments was used to test the variation of promoter strength (RPU) in different concentration of arabinose. Each strain of cells inoculated overnight in various arabinose concentration above. The cells were diluted around 10 fold and grown until they reached mid-log phase (OD600 0.3-0.5). Cells were fixed and fluorescence was measured using FACS. The graph represent triplicate mean ±SD. </h6> <br><br><br />
<br />
<p><b><u>RPU of PBAD in different cell strains</u></b><br><br />
We thought that Relative Promoter Unit (RPU) defined by Endy et al. would be a better a measure of promoter strength at different arabinose concentration than simply comparing the fluorescence measurement of different strains. This is because simply measuring and comparing fluorescence as an output can also be affected by the concentration of cells. For example, having high concentration of low GFP expressing cells can rise to high fluorescence output. Also, fluorescence output have a lot of variability depending on the experimental context. Because RPU is a ratio to fluorescence measurement, the effect mentioned above can be minimized (See Methods for RPU calculation).<br><br><br />
<br />
We observed clearly a different promoter response between other two cell types and DH5α. The graph of DH10B and BW25113, however, overlapped and therefore t-test was conducted to statistically identify the significance difference. The P-value was 0.1207. So if we use the 0.05 confidence level, we cannot statistically prove that the two curves are different. <br><br><br />
<br />
The lower RPU of DH5α was expected because DH5α has araC gene, gene for the repressor, in its genome. This could have caused the lower promoter strength of PBAD¬ in DH5α.<br><br><br />
<br />
For DH10B and BW25113 we see a very clear “all-or-none” response. The RPU reaches a plateau at 0.2% of arabinose. For DH5α, it is less obvious, because the initial increase of RPU at 0.2% arabinose is around 0.1, lower than the other two strains. With the given graph, statically, we cannot say that the RPU level gradually increases as % arabinose increases. All in all, all three strains produced all-or-none response. <br><br><br />
<br />
<p><b><u>P<sub>BAD</sub> Leakage</u></b><br><br />
Leakage was observed for PBAD promoter ¬in DH10B. Although the RPU was relatively higher compared to that of other two strains, DH10B showed leakage at no arabinose induction. The fluorescence measurement normalized to the maximum observed fluorescence (for DH10B) was around 0.12 which is quite significant if we want to have a non-leaky system. For BW25113 and DH5α the leakages were statistically insignificant and close to normalized fluorescence value of 0. The low leakage in DH5α, once again, could be explained by the additional copy of araC in the genome. The low leakage BW25113, however, is harder to explain. Nonetheless, if we want a low-leakage system that can reach relatively high RPU upon arabinose induction, out of the three strain we have used, BW25113 would be the best option. <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/5/54/Pbad_RPU_2.png" /><br />
<h5 style="font-size: 13px">Figure 4. Leakage of Pbad promoter in different cell strain. </h5><br />
<h6 style= "font-size: 13px"> For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD. </h6> <br><br><br />
<br />
<p><b><u>3-D graphs for DH10B and DH5α</u></b><br><br />
We also tried to replicate the experiment by Groningen 2011 by producing a 3-D graph. We, however, made some deviation. At 3’ of the PBAD promoter, instead of using BBa_E0840 (GFP generator), we used BBa_E0240 (GFP generator). We also could not take measurements every 15 minutes interval because we had to measure the fluorescence at each time point manually. Fluorescence was instead measured every two hours. Lastly, OD600 was also monitored and graphed (Figure 5.) <br><br><br />
<br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /><br />
<h5 style="font-size: 13px">Figure 5. </h5><br />
<h6 style= "font-size: 13px"> </h6> <br><br><br />
<br />
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<div class='content_1'><h3>Discussion</h3><br />
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<tr class= "content_row"><br />
<td class= "content_cell"><br />
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<p>Groningen 2011 results may not truly represent the gradual induction of PBAD promoter <br><br><br />
<br />
We believe that it is actually difficult to analyze the promoter’s gradual or all-or-none response looking the 3-dimensional graph that Groningen 2011 team presented. The 3 dimensional graph has three parameters: time, various arabinose concentrations and fluorescence. The graph does not consider the OD600 value which can represent the concentration of cells. If the growth rate of the cells are different in different arabinose concentration, the final concentration of cells at given point of time can vary. Because the fluorimetry measurements, the method that Groningen 2011 used, measures the fluorescence of the entire population of cells, the fluorescence can be affected by the concentration of cells and hence show a response that is not all-or-none. Looking at Groningen’s results, it would be more appropriate to say that the population of cells that is transformed with plasmid containing BBa_K607036 (BBa_I0500-BBaE0840) show a gradual response to increasing arabinose concentration. <br><br><br />
<br />
</p><br />
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<br><br />
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<div class='content_1'><h3>Methods</h3><br />
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<p>For each cell strains, we first transformed with three different plasmids: pSB3K3-BBa_E0240, pSB3K3-BBa_I20260, and pSB3K3-BBa_I0500-BBa_E0240. We grew the cells overnight in various arabinose concentrations (%w/v) from 0% to 1.0% with 0.2% increments. The cells were diluted the next around 10 fold and grown again until they reached mid-log phase (OD600 0.4-0.5). Cells were fixed and fluorescence was measured using FACS. RPU was calculated first subtracting the autofluorescence (fluorescence of cells with pSB3K3-BBa_E0240, and dividing the fluorescence of cells containing pSB3K3-BBa_I0500-BBa_E0240 with cells containing pSB3K3-BBa_I20260.<br><br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/6/60/Riboregulator_ust_Equation.png"/> <br><br><br />
<br />
For simplicity, we assumed the growth rate of the cells transformed with pSB3K3-BBa_I0500-BBa_E0240 and cells transformed with pSB3K3-BBa_I20260 had same similar growth rate. <br />
<br />
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<div></div>Jordyevanhttp://2014.igem.org/File:3dgraph_pbad.pngFile:3dgraph pbad.png2014-10-17T20:27:59Z<p>Jordyevan: </p>
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<div></div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T20:11:37Z<p>Jordyevan: </p>
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<br><h2>Riboregulator Characterization</h2><br><br />
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<div class='content_1'><h3>Introduction</h3><br />
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<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
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<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 (BBa_J01080) Key3 (BBa_J01086)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock (BBa_K175031)Key for medium lock (BBa_K175032)</td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
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<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
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<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter (BBa_J23102) to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible PBAD promoter (BBa_I0500)<br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
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<br><h2>PBAD Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
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<p>PBAD promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the PBAD promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the PBAD promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the PBAD promoter.</p><br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/modulesTeam:Hong Kong HKUST/pneumosensor/modules2014-10-17T19:37:57Z<p>Jordyevan: </p>
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<h2>Pneumosensor Module Description</h2><br />
<p style= "font-size:30px; text-align:center"><u><br>Overview</u></p><br />
<p> Pneumosensor primarily adopts the quorum sensing pathway components in <i>Streptococcus pneumoniae</i> to detect populations of <i>S. pneumoniae</i>. The main <br />
advantage of this system is its detection specificity- the Gram-positive quorum sensing mechanism is incorporated into the Gram-negative bacteria <i>E. coli</i> to <br />
eliminate possible cross-talk of the autoinducer molecule, competence-stimulating peptide (CSP) with native <i>E. coli</i> molecules. <br />
<br><br><br />
There are two modules to our Pneumosensor- the Detection Module and the <i>S. pneumoniae</i> σ<sup>x</sup> Promoters Module. The Detection Module comprises of the CSP <br />
receptor ComD, its response regulator ComE and the promoter P<sub>comCDE</sub> which is induced by phosphorylated ComE. The σ<sup>x</sup> Promoters Module involves a <br />
highly specific reporting system, whereby σ<sup>x</sup> is associated with RNA polymerase and binds to promoters P<sub>celA</sub> and P<sub>comFA</sub> which <br />
are specific to σ<sup>x</sup> for activation. These promoters then drive the expression of GFP as a reporting system. Another protein, ComW, is expressed alongside the <br />
σ<sup>x</sup> as its stabilizer against proteolysis. </p><br><br><br />
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<img src="https://static.igem.org/mediawiki/2014/thumb/5/5d/Module_2HKUST.png/610px-Module_2HKUST.png"/> <p><i>S. pneumoniae</i> &sigma;<sup>x</sup> promoters Module</p><br />
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<div class='content_1' id="1"><h3>Detection Module Description </h3><br />
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<h6><b>Figure 1. Detection Module Diagram</h6></b><br><br />
<h7>CSP released by <i>S. pneumoniae</i> is detected by its receptor ComD, which autophosphorylates at the expenditure of ATP. ComD~P then phosphorylates <br />
ComE. ComE~P then induces the promoter P<sub>comCDE</sub>, which drives the expression of GFP.</h7><br />
</div><br />
</div><br />
<p class="first_letter_enhanced">Transformation in <i>Streptococcus pneumoniae</i>, like many other species, depends on specialized state called competence. <br />
Competence is achieved during the exponential growth stage of pneumococcal culture by the secretion of competence-stimulating peptide (CSP), which is a 17-<br />
residue long pheromone that is species-specific. Our team aims to adopt the specific CSP sensing mechanism of <i>S. pneumoniae</i> to detect its populations.<br />
</p><br />
<br><br />
<p>The mechanism we adopted is controlled by a two-component regulatory system (TCS), which consists of the histidine kinase (HK) ComD and its <br />
cognate response regulator (RR) ComE (<a href="http://parts.igem.org/Part:BBa_K1379051">BBa_K1379051</a>). When CSP binds to it, ComD autophosphorylates to become phospho-ComD, ComD~P; at the expenditure of ATP. <br />
The cytosolic protein ComE is then phosphorylated by ComD~P through transphosphorylation reactions, producing ComE~P. Genes coding for ComD and ComE are constitutively <br />
expressed in our Pneumosensor.<br />
</p><br />
<br><br />
<p>ComE~P binds to repeat sites adjacent to the <i>comCDE</i> and <i>comAB</i> operons (Ween et al., 1999), creating a positive <br />
feedback loop, producing both pre-CSP and its required machinery for maturation and transport. The signal is thus amplified and competence is <br />
coordinated throughout the population. <br />
</p><br />
<br><br />
<p>As part of our detection circuit design, we make use of the inducible promoter from the <i>comCDE</i> operon, P<sub>comCDE</sub>. We obtain the sequence by oligos, <br />
and will characterize it using green fluorescence protein (GFP) that we constructed the downstream of the promoter by BioBrick RFC10. Rather than using ComE~P that has to<br />
be phosphorylated by ComD~P, which involves a chain of reactions, we use a phosphorylmimetic ComE mutant, ComE<sup>D58E</sup>, kindly<br />
shared with us by Bernard Martin et al. After characterization of P<sub>comCDE</sub>, we hope to put P<sub>comCDE</sub> together with the <i>S. pneumoniae</i> σ<sup>x</sup> <br />
promoters module, for the ultimate goal of creating a tightly regulated automatic detection of <i>S. pneumoniae</i>.<br />
</p><br />
<br />
</div><br />
</td><br />
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</table><br />
</div><br />
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<br />
<div class='content_1' id="2"><h3><i>S. pneumoniae</i> σ<sup>x</sup> promoters Module Description</h3><br />
<br />
<h2><center><b><u>&sigma;<sup>x</sup> - Com-Box mechanism</u></b></center></h2><br />
<br />
<table class="content_table" align= "center" ><br />
<tr class= "content_row" valign= "top"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row" ><br />
<div class= "embedded_image_left"><br />
<div class="content_image"><br />
<img src= "https://static.igem.org/mediawiki/2014/d/d0/Sigmax_combox_final.png"/><br />
<h6><b>Figure 1. &sigma;<sup>x</sup>-Com-Box promoter mechanism</h6></b><br><br />
<h7> The reporter system contains a constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a>, which continuously expresses &sigma;<sup>x</sup> required for Com-Box promoter induction. &sigma;<sup>x</sup> will then bind to Com-Box promoter and express green fluorescence protein. The whole construct was built in <i>E. coli</i> DH10B strain. </h7><br />
</div><br />
</div><br />
<p class="first_letter_enhanced">In order to achieve the functionality of pneumosensor, we must have a highly specific reporting system which will only give fluorescent signal under the presence of <i>S. pneumoniae</i>. In search for the suitable gene circuit, the discovery by Prof. Morrison on the competence for genetic transformation in <i>S. pneumoniae</i> which depends on quorum-sensing system to control many competence-specific genes acting in DNA uptake, processing, and integration has provided the ideal framework for this module. (Lee and Morrison, 1999) There is a link between this quorum-sensing system and the competence-specific genes, which is an alternative &sigma;<sup>x</sup> (ComX protein) that serves as a competence-specific global transcription modulator. (Luo and Morrison, 2003) In <i>S. pneumoniae</i>, competence (a state capable of being genetic transformed) happens transiently during the log phase growth, and is regulated by a quorum sensing system utilizing the competence-stimulating peptide (CSP). Upon stimulation by CSP, &sigma;<sup>x</sup> will be expressed and associated with RNA polymerase apoenzyme. The resulting holoenzyme will then be guided by &sigma;<sup>x</sup> to initiate transcription of a set of “late” genes enabling genetic transformation and other unknown functions. Characterized genes regulated by &sigma;<sup>x</sup> were found to contain an 8 base pairs consensus sequence TACGAATA known as the Cin-Box or the Com-Box. (Piotrowski, Luo, & Morrison, 2009). Taking advantage of this competence-specific mechanism, it is now able to produce the <i>S. pneumoniae</i> sensing device of high specificity by incorporating this system into <i>E. coli</i>. <br><br />
<br />
</p><br />
<br><br />
<p><br />
iGEM 2014 Hong_Kong_HKUST Team has cloned &sigma;<sup>x</sup> from <i>S. pneumoniae</i> strain NCTC7465 and characterized its ability to initiate transcription of two downstream promoters with different lengths: P<sub>celA</sub> (<a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>) and P<sub>comFA</sub> (<a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>), which have the consensus Com-Box sequence. Though much information about the promoters is readily available nowadays, its characterization of promoter activity, specificity, sequence, as well as the biomolecular mechanism can be greatly enhanced with further investigations and experiments. Hence, we were interested in reproducing this gene circuit with all the associated genes and promoters to be combined into a single transcriptional unit. Despite the suggested susceptibility to leakage and other factors that may hinder or interrupt the mechanism, researches have reported that the pathway was highly specific to certain environmental conditions and stress, suggesting minimal or no leakage in the entire process. <br><br />
<br><br />
<p><br />
P<sub>celA</sub> and P<sub>comFA</sub> promoters have high specificity to &sigma;<sup>x</sup> for activation, so genes downstream the promoters will be translated only if &sigma;<sup>x</sup> is present. Hence, by using fluorescence protein as a reporting mechanism, this &sigma;<sup>x</sup>, P<sub>celA</sub> and P<sub>comFA</sub> promoters system could be further utilized as a specific reporter device in <i>E. coli</i> DH10B strain that could be used by iGEM communities.<br />
</p> <br />
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<div class='content_1'><h2><center><b><u>&sigma;<sup>x</sup>-ComW mechanism</u></b></center></h2><br />
<table class="content_table" align= "center" ><br />
<tr class= "content_row" valign= "top"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class= "embedded_image_right"><br />
<div class="content_image"><br />
<img src= "https://static.igem.org/mediawiki/2014/e/e4/Wiki_interactioncomW_copy.png"/><br />
<h6><b>Figure 2. &sigma;<sup>x</sup> - <i>comW</i> Interaction Diagram</h6></b><br><br />
<h7>&sigma;<sup>x</sup> and ComW protein are both produced by a constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a>, which continuously expresses &sigma;<sup>x</sup> required for P<i><sub>celA</sub></i> and P<i><sub>comFA</sub></i> promoters induction, and ComW protein is required for &sigma;<sup>x</sup> stabilization. ComW protein acts as a barrier that protects &sigma;<sup>x</sup> from being degraded by ClpXP degradation enzyme, hence it increases the production of &sigma;<sup>x</sup>. The increase in &sigma;<sup>x</sup> production will increase the expression of green fluorescence protein by P<i><sub>celA</sub></i> and P<i><sub>comFA</sub></i> promoters.</h7><br />
</div><br />
</div><br />
<p class="first_letter_enhanced">To complete the story of competence regulation mechanism from <i>S. Pneumoniae</i>, we would also like to integrate another positive factor involved in competence regulation which was later found out to be ComW. Prof. Morrison's lab released another research paper on the identification of a new component in the regulation of genetic transformation in <i>S. Pneumoniae</i>. The gene <i>comW</i> (SP0018) was found to be <br />
regulated by the quorum-sensing system and is required for a high-level of competence (Luo, Li, and Morrison, 2004). Coexpression of ComW with &sigma;<sup>x</sup> restores the accumulation of &sigma;<sup>x</sup> and the expression of late genes as ComW contributes to the stabilization of the alternative sigma factor X against proteolysis by ClpXP and is required for full activity of &sigma;<sup>x</sup> in directing transcription of late competence genes (Piotrowski, Luo, and Morrison, 2009). <br />
</p><br />
<br><br />
<p><br />
Based on these findings, we tried to integrate this ComW into the mechanism to see whether and how the presence of ComW affects &sigma;<sup>x</sup>. We firstly cloned out the <i>comX</i> gene expressing &sigma;<sup>x</sup>, and <i>comW</i> genes from the genomic DNA of <i>S. pneumoniae</i> NCTC 7465 strain. We then used <a href= "http://parts.igem.org/Part:BBa_K880005">BBa_K880005</a> (consisting of constitutive promoter <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_J23100">BBa_J23100</a> and strong RBS <a href= "http://parts.igem.org/wiki/index.php?title=Part:BBa_B0034">BBa_B0034</a>) from the BioBricks to express those genes.<br><br><br />
</p><br />
<br />
<br />
<br />
</div><br />
</td><br />
</tr><br />
</div><br />
</table><br />
</div><br />
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<br />
<br />
<br><br><br />
<div class='content_1'><br />
<table class="content_table" align= "center" ><br />
<tr class= "content_row" ><br />
<td class= "content_cell" colspan= "2"><br />
<div class= "content_area_two_row"><br />
<p><br />
<u>References</u><br />
<br><br />
<br><br />
B. Martin et al &quot;ComE/ComE~P interplay dictates activation or extinction status of pneumococcal X-state (competence) &quot; 2012<br />
<br><br />
<br><br />
B. Martin et al &quot;Cross-regulation of competence pheromone production and export in the early control of transformation in <i>Streptococcus pneumoniae</i> &quot; 2000<br />
<br><br />
<br><br />
B. Martin et al &quot;Expression and maintenance of ComD–ComE, the two-component signal-transduction system that controls competence of <i>Streptococcus pneumoniae</i> mmi_7071 1513..1528 &quot; <br />
<br><br />
<br><br />
Cheng, Q., Campbell, E.A., Naughton, A.M., Johnson, S., and Masure, H.R. (1997) The com locus controls genetic transformation in <i>Streptococcus pneumoniae</i>. Mol Micro-biol 23: 683±692.<br />
<br><br />
<br><br />
Pestova, E.V., and Morrison, D.A. (1998) Isolation and characterization of three <i>Streptococcus pneumoniae</i> transformation-specific loci by use of a lacZ reporter insertion vector. J Bacteriol 180: 2701±2710.<br />
<br><br />
<br><br />
Ween, O., Gaustad, P., and Havarstein, L.S. (1999) Identification of DNA binding sites for ComE, a key regulator of natural competence in <i>Streptococcus pneumoniae</i>. Mol Microbiol 33: 817±827.<br />
<br><br />
<br><br />
A. Piotrowski, P. Luo, & D. A. Morrison. (2009). Competence for Genetic Transformation in <i>Streptococcus pneumoniae</i>: Termination of Activity of the Alternative Sigma Factor ComX Is Independent of Proteolysis of ComX and ComW. <i>Journal of Bacteriology</i>, 191(10), 3359-3366. doi:10.1128/JB.01750-08<br />
<br><br />
<br><br />
P. Luo & D. A. Morisson. (2003). Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in <i>Streptococcus pneumoniae</i>. <i>Journal of Bacteriology</i>, 185(1), 349-358. doi: 10.1128/JB.185.1.349-358.2003<br />
<br><br />
<br><br />
C. K. Sung & D. A. Morrison. (2005). Two Distinct Functions of ComW in Stabilization and Activation of the Alternative Sigma Factor ComX in <i>Streptococcus pneumoniae</i>. <i>Journal of Bacteriology</i>, 185(9), 3052-3061. doi: 10.1128/JB.187.9.3052-3061.2005<br />
<br><br />
<br><br />
P. Luo, H. Li, & D. A. Morrison. (2004). Identification of ComW as a new component in the regulation of genetic transformation in <i>Streptococcus pneumoniae</i>. <i>Molecular Microbiology</i>, 54(1), 172-183. doi: 10.1111/j.1365-2958.2004.04254.x<br />
<br><br />
<br><br />
M. S. Lee & D. A . Morrison. (1999). Identification of a New Regulator in <i>Streptococcus pneumoniae</i> Linking Quorum Sensing to Competence for Genetic Transformation. <i>Journal of Bacteriology</i>, 181(16), 5004-5016.<br />
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<div></div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/partsTeam:Hong Kong HKUST/pneumosensor/parts2014-10-17T18:19:20Z<p>Jordyevan: </p>
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<h2>Pneumosensor Parts</h2><br />
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<div class = "catalog_table_area"><br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name</th><br />
<th scope="col">Type</th><br />
<th scope="col">Description</th><br />
<th scope="col">Length</th><br />
</tr><br />
</thead><br />
<tbody><br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379004">BBa_K1379004</a></td> <br />
<td>Coding</td> <br />
<td>σ<sup>x</sup></td> <br />
<td>513</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a></td> <br />
<td>Generator</td> <br />
<td>σ<sup>x</sup> Generator</td> <br />
<td>711</td><br />
</tr> <br />
<br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a></td> <br />
<td>Measurement</td> <br />
<td>P<sub>celA</sub>-E0240 (Measurement Kit for P<sub>celA</sub>)</td> <br />
<td>984</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a></td> <br />
<td>Measurement</td> <br />
<td>P<sub>comFA</sub>-E0240 (Measurement Kit for P<sub>comFA</sub>)</td> <br />
<td>1044</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379045">BBa_K1379045</a></td> <br />
<td>Composite</td> <br />
<td>σ<sup>x</sup> + B0015</td> <br />
<td>650</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a></td> <br />
<td>Regulatory</td> <br />
<td>P<sub>celA</sub> 100 basepairs combox promoter</td> <br />
<td>100</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a></td> <br />
<td>Regulatory</sub></td> <br />
<td>P<sub>comFA</sub> 160 base pairs combox promoter</td> <br />
<td>160</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a></td> <br />
<td>Measurement</td> <br />
<td>σ<sup>x</sup> + P<sub>celA</sub>-E0240</td> <br />
<td>1703</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a></td> <br />
<td>Measurement</td> <br />
<td>σ<sup>x</sup> + P<sub>comFA</sub>-E0240</td> <br />
<td>1763</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379051">BBa_K1379051</a></td> <br />
<td>Coding</td> <br />
<td><i>comE</i></td> <br />
<td>753</td><br />
</tr> <br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/partsTeam:Hong Kong HKUST/pneumosensor/parts2014-10-17T18:18:31Z<p>Jordyevan: </p>
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<div class = "catalog_table_area"><br />
<table class= "catalog_table"><br />
<thead><br />
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<th scope="col">Name</th><br />
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<th scope="col">Description</th><br />
<th scope="col">Length</th><br />
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</thead><br />
<tbody><br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379004">BBa_K1379004</a></td> <br />
<td>Coding</td> <br />
<td>σ<sup>x</sup></td> <br />
<td>513</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a></td> <br />
<td>Generator</td> <br />
<td>σ<sup>x</sup> Generator</td> <br />
<td>711</td><br />
</tr> <br />
<br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a></td> <br />
<td>Generator</td> <br />
<td>P<sub>celA</sub>-E0240 (Measurement Kit for P<sub>celA</sub>)</td> <br />
<td>984</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a></td> <br />
<td>Generator</td> <br />
<td>P<sub>comFA</sub>-E0240 (Measurement Kit for P<sub>comFA</sub>)</td> <br />
<td>1044</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379045">BBa_K1379045</a></td> <br />
<td>Composite</td> <br />
<td>σ<sup>x</sup> + B0015</td> <br />
<td>650</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a></td> <br />
<td>Regulatory</td> <br />
<td>P<sub>celA</sub> 100 basepairs combox promoter</td> <br />
<td>100</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a></td> <br />
<td>Regulatory</sub></td> <br />
<td>P<sub>comFA</sub> 160 base pairs combox promoter</td> <br />
<td>160</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a></td> <br />
<td>Measurement</td> <br />
<td>σ<sup>x</sup> + P<sub>celA</sub>-E0240</td> <br />
<td>1703</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a></td> <br />
<td>Measurement</td> <br />
<td>σ<sup>x</sup> + P<sub>comFA</sub>-E0240</td> <br />
<td>1763</td><br />
</tr> <br />
<br />
<tr><br />
<td scope="row"><a href="http://parts.igem.org/Part:BBa_K1379051">BBa_K1379051</a></td> <br />
<td>Coding</td> <br />
<td><i>comE</i></td> <br />
<td>753</td><br />
</tr> <br />
<br />
</tbody><br />
</table><br />
<br />
</div><br />
</div><br />
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</body><br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T17:04:34Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
<html><head><br />
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<br><h2>Riboregulator Characterization</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br />
<br />
</a></div><br />
<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 (BBa_J01080) Key3 (BBa_J01086)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock (BBa_K175031)Key for medium lock (BBa_K175032)</td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src=""><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter (BBa_J23102) to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible PBAD promoter (BBa_I0500)<br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<!-- end of one row of content , two column--><br />
<br />
</div><br />
</div><br />
<br><br><br />
<div id="content_container"><br />
<!-- one row of content , two column one picture right--><br />
<div id="description_area"><br />
<br><h2>PBAD Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>PBAD promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the PBAD promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the PBAD promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the PBAD promoter.</p><br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/resultsTeam:Hong Kong HKUST/riboregulator/results2014-10-17T17:04:18Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
<html><head><br />
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|<br />
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<div id="content_container"><br />
<br />
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<br><h2>Riboregulator Results</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Overview</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>Although there is a significant number of regulatory RNAs available in the registry, a comprehensive characterization information that the iGEM community can <br />
use to compare and contrast different regulatory RNAs (especially CR-TA riboregulators) is missing.Therefore we wanted to provide characterization information of<br />
regulatory RNAs so teams and labs will be confident in using these devices. </p><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<!-- end of one row of content , two column--><br />
<br />
<!-- one row of content , two column one picture right--><br />
<div class='content_1'><h3>Construct</h3><br />
<table class="content_table" align= "center" ><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>In order to provide reliable characterization data which will help iGEM teams to identify which CR-TA riboregulator works best for their projects. We have built <br />
constructs like the diagram provided below:<br />
(provide diagram)<br />
<p>Cis-repressing part which was generated by using the Lock and Key algorithm generator from TU Delft 2009 iGEM team and followed by de novo synthesis from oligo <br />
annealing. After annealing, these dsDNA was directly inserted into the vector containing <a href="http://parts.igem.org/Part:BBa_I13401">BBa_I13401</a> ( GFP reporter for RHS of library test constructs). A promoter, <br />
<a href="http://parts.igem.org/Part:BBa_J23102">BBa_J23102</a> was placed upstream by the scar formation of cis-repressing part therefore cis-repressing part can be <br />
constitutively transcribed.Trans-activating part was produced by the same method as cis-repressing part but an inducible promoter, P<sub>bad</sub>, <a href="http://parts.igem.org/Part:BBa_I13401">BBa_I0500 </a>and double terminator, <br />
<a href="http://parts.igem.org/Part:BBa_BBa_B0015">BBa_B0015 </a>were used to replace <a href="http://parts.igem.org/Part:BBa_I13401">BBa_J23102 </a>and <br />
<a href="http://parts.igem.org/Part:BBa_I13401">BBa_I13401 </a>respectively. A low copy plasmid, pSB3K3 was used for GFP expression measurements. For the control of our characterization, construct containing only RBS, <br />
instead of CR and another construct missing TA were built. To obtain the fluorescence level, autofluorescence had to be subtracted so bacteria containing pSB3K3-E0240<br />
plasmid were used. We have submitted Cis-repressing and Trans-activating parts as well as some of intermediary parts. </p><br />
</div><br />
</td><br />
<br />
</tr><br />
</table><br />
</div><br />
<!-- end of one row of content , two column--><br />
<br><br><br />
<br />
<!-- one row of content , two column one picture left--><br />
<div class='content_1'><h3> Results</h3><br />
<table class="content_table" align= "center" ><br />
<br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="content_image"><br />
<br />
</div><br />
<p><br />
<img style= "width:50%" src= "#"/><br />
<br><br />
The above bar graphs represents the level of fluorescence measured by ? and it was plotted as fluorescence/OD600 on y-axis. Culture containing bacteria was <br />
incubated for X hours. (From left to right) For controls of each set of an experiment, (1) a construct without trans-activating part (2) a construct without<br />
cis-repressing part but with a ribosomal binding site (3) a construct without both cis-repressing and trans-activating parts. For our experiment, a construct <br />
containing both cis-repressing and trans-activating parts was used. arabinose concentration of 0%, 1% and 2.5% were used to induce the inducible promoter, P<sub>bad</sub><br />
(<a href="https://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterization">P<sub>bad</sub> characterization</a>). </p><br />
<br />
<br><br><br />
<img style= "width:50%" src= "#"/><br />
<br><br />
<p>Furthermore the orthogonality of five different sets of riboregulators were tested in parallel to the repression and activation strength of riboregulators. <br />
The increase in fluorescence level was calculated using the fluorescence level obtained by plate reader at 0% and 2.5% of arabinose concentrations.</p><br />
</div><br />
</td><br />
<br />
<br />
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</table><br />
</div><br />
<br />
<div class='content_1'><h3> Discussion</h3><br />
<table class="content_table" align= "center" ><br />
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<td class= "content_cell"><br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/riboregulator/characterizationTeam:Hong Kong HKUST/riboregulator/characterization2014-10-17T16:44:06Z<p>Jordyevan: </p>
<hr />
<div>{{Team:Hong_Kong_HKUST/shell|<br />
<html><head><br />
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<br><h2>Riboregulator Characterisation</h2><br><br />
</div><br />
<br />
<br />
<br />
<!-- one row of content , two column--><br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<div class="ienlarger"><a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br />
<a href="#nogo"><img src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png" /><br />
<br />
<br />
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<br />
<p>Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system, <br />
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.<br />
When the transcript is formed, the cis-repressing sequence can form a loop to form a complementary base pairs with the RBS and blocking the ribosome entry <br />
to RBS. CrRNA is commonly called “lock” because it “locks” the translation of proteins. When there is a lock, we need a “key”. Component of the system that<br />
act as a key is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore activate translation. The HKUST iGEM 2014 <br />
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team. <br />
<br />
<br>Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:<br><br />
<br />
<table class= "catalog_table"><br />
<thead><br />
<tr><br />
<th scope="col">Name and registry code</th><br />
<th scope="col">Group</th><br />
<th scope="col">Cognate pair</th><br />
<br />
</tr><br />
</thead><br />
<tbody><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 3 (BBa_J01080) Key3 (BBa_J01086)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Medium lock (BBa_K175031)Key for medium lock (BBa_K175032)</td> <br />
<td>iGEM09_TUDelft</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<tr><br />
<td scope="row">Lock 1 (BBa_J01010) Key 1 (BBa_J01008)</td> <br />
<td>iGEM 2005_Berkeley (Golden Bear)</td> <br />
<td>Yes</td> <br />
</tr><br />
<br />
<br />
</table><br />
<p>Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that <br />
Lock 3c and Key 3c (Table 1.) were cognate pairs, but they turned out to be that iGEM 2006_Berekley simply made different variants of Lock 3 and Key 3. They gave put an <br />
alphabet at the end of the name every time they produced different variant of lock 3 and key 3. The lock 3 and key 3 variants were created independently from each other so <br />
the letters at the end of name does not mean correspondence. Other teams should take note of this when they consider using riboregulators variants from iGEM 2006_Berkeley.</p><br />
<br><br />
</div></div><br />
</td><br />
</tr><br />
</table><br />
<br />
<br />
<div class='content_1'><h3>Riboregulator Results</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<img src=""><br />
<br><br />
<p>To characterize the different riboregulator pairs, we kept the genetic context identical except for the various cr-repressing sequence, trans-activating sequence and the <br />
RBS. The RBS sequence also had to be different for some of the riboregulator system. This is because the cr-repressing sequence depends on the RBS sequence; In order to <br />
repress translation, the cis-repressing sequence need to interact with the RBS, and the interaction depends on the sequences. Since different teams used different RBS to <br />
design their cis-repressing sequence, we also had to use corresponding RBS for characterization. We had a constitutive promoter (BBa_J23102) to drive the expression of the <br />
cis-repressed GFP translation unit. For the expression taRNA, we wanted to control the expression and therefore we decided to use arabinose inducible PBAD promoter (BBa_I0500)<br />
. The promoter was chosen because the 3’ end after the transcription start site of the promoter is short. Longer 3’ end can affect the function of the taRNA (source)<br />
(Figure 1. A). (fsd different between –TA and -CR). </p><br />
<br><br><br />
<p>For the riboregulator system to work, the repression of GFP synthesis needs to be first observed when the cis-repressing sequence is added 5’ of the RBS of the system. <br />
Significant repression can be seen in Lock 1-Key1, Lock 3-Key 3, and Medium lock (Lock m)-Key for medium lock (Key m) cognate pairs (Figure 1. B, C, D respectively). <br />
For Lock 3c-Key 3c pair, we do not see repression when cis-repressing sequence is introduced to the system. Instead, converse can be observed. When we don’t have <br />
cis-repressing sequence, we see significant drop in the fluorescence (Figure 1.E). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system <br />
seems very leaky compared to other riboregulator system. Also, no fluoresce can be observed when trans-activating component is introduced without the presence of the <br />
cis-repressing sequence (Figure 1.F). For the sake of time, we did not have the chance to sequence confirm the entire set of riboregulator pairs’ controls. We did, however, <br />
sequence verified the cognate pairs and lock3c-key 3c pair. The sequence matched except for HKUST lock 1-HKUST key 1. The deviation from expected result for HKUST lock <br />
1-HKUST key 1 may be explained by the sequencing results. For lock3c-key3c, since the sequence matched 100%, we can simply conclude that we have a faulty system.<br />
After repression, the system needs to be activated when taRNA is expressed. </p><br />
</div><br />
</td><br />
</tr><br />
</table><br />
</div><br />
<!-- end of one row of content , two column--><br />
<br />
</div><br />
</div><br />
<br><br><br />
<div id="content_container"><br />
<!-- one row of content , two column one picture right--><br />
<div id="description_area"><br />
<br><h2>PBAD Characterization</h2><br><br />
</div><br />
<br />
<div class='content_1'><h3>Introduction</h3><br />
<table class="content_table" align= "center" valign= "top"><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"><br />
<p>PBAD promoter is an arabinose inducible promoter. In nature, the promoter exist in the arabinose operon to regulate the transcription of araB, araA, and araD.<br />
The arabinose operon or the ara operon encode enzymes needed or the catabolism of arabinose to xylulose 5- phosphate which is an intermediate of the pentose phosphate <br />
pathway. The Pc promoter which is adjacent to the PBAD promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity<br />
of the PBAD promoter when arabinose is absent. Once arabinose is present,the AraC protein binds to the arabinose and dimerize. The dimerize form of AraC-arabinose can <br />
activate the PBAD promoter.</p><br />
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<h2>Pneumosensor Characterization</h2><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379004"> σ<sup>x</sup>(BBa_K1379004) </a></h3><br />
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<p><br><b><u>Introduction</u></b> <br><br><br />
<p class="first_letter_enhanced">To test the functionality of &sigma;<sup>X</sup>, we first enable constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator, <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>.The generator was then assembled with the standard promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, with either promoter P<sub>celA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a>) and P<sub>comFA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a>). <i>E. coli</i> colonies holding the resulting constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, which is <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the general negative control. for background fluorescence. Measurement kits for P<sub>celA</sub> P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used as negative controls for function of &sigma;<sup>X</sup>.<br />
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<p><br><b><u>Results</u></b> <br><br></p><br />
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<h5 style="font-size: 13px">Figure 1. P<sub>celA</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.</h5><br />
<h6 style= "font-size: 13px"> Only in the presence of &sigma;<sup>X</sup> would P<sub>celA</sub> and P<sub>comFA</sub> be turned on, as GFP expression could be seen when &sigma;<sup>X</sup> present. Therefore, &sigma;<sup>X</sup> is functional. P<sub>celA</sub> and P<sub>comFA</sub> gave little GFP signal in the absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter BBa_J23101 in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.</h6><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379000">P<sub>celA</sub> (BBa_K1379000)</a> and <a href= "http://parts.igem.org/Part:BBa_K1379001">P<sub>comFA</sub> (BBa_K1379001) </a></h3><br />
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<p class="first_letter_enhanced"> <br><br>For characterization, P<sub>celA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>celA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a>. <br><br><br />
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P<sub>comFA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>comFA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a>. <br><br><br />
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Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>celA</sub> and P<sub>comFA</sub> Measurement Kits with and without the &sigma;<sup>X</sup> generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the negative control for background fluorescence. <br><br><br />
<br />
Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). <i>E. coli</i> DH10B strains holding the constructs with or without &sigma;<sup>X</sup> generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> measurement device <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was measured alongside. The measurement was done with 3 replicas. <br />
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<h5 style="font-size: 13px">Figure 2. P<sub>celA</sub> has 0.53 RPU and P<sub>comFA</sub> hsa 1.21 RPU when paired with &sigma;<sup>X</sup> generator.</h5><br />
<h6 style= "font-size: 13px"> P<sub>celA</sub> and P<sub>comFA</sub> was measured in reference to <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> constitutive promoter with and without &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. RPU shown was calculated from 3 replicas.</h6> <br><br><br />
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<div class='content_1'><h3>Characterization Method</h3><br />
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<p><u><b>Construction</b></u><br><br><br />
1. Construct σ<sup>x</sup> Generator pSB3K3-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br />
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Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a> to pSB3k3.<br><br><br />
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2. Construct σ<sup>x</sup> Generator pSB3K3-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> <br><br />
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Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a> to pSB3K3.<br><br><br />
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3. Transforming pSB3K3-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
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4. Transforming pSB3K3-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
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5. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit<br><br><br />
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6. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit.<br><br><br />
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<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
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2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on procedure number 1. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
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3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium in the 96 Deep Well plate. <br><br><br />
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4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
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5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
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6. Measuring the GFP intensity and OD595 values (we used Envision Multilabel Reader) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
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Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
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- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
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7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
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<p><br><u><b>Data Processing</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
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3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
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4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
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5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
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6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
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<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
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<u><b>References</b></u><br><br></p><br />
<p><br />
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BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 and http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 <br />
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Luo P., & Morrison D. (2003).<i> Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae</i>. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003<br />
<br><br><br />
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Piotrowski A., Luo P., & Morrison D. (2009). <i>Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.</i> Journal of Bacteriology. doi:10.1128/JB.01750-08<br />
<br><br><br />
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Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). <i>Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.</i> Molecular Systhetic Biology .doi:10.1038/msb.2013.58<br />
<br><br><br />
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J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterizationTeam:Hong Kong HKUST/pneumosensor/characterization2014-10-17T16:32:07Z<p>Jordyevan: </p>
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<h2>Pneumosensor Characterization</h2><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379004"> σ<sup>x</sup>(BBa_K1379004) </a></h3><br />
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<p><br><b><u>Introduction</u></b> <br><br><br />
<p class="first_letter_enhanced">To test the functionality of &sigma;<sup>X</sup>, we first enable constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator, <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>.The generator was then assembled with the standard promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, with either promoter P<sub>celA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a>) and P<sub>comFA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a>). <i>E. coli</i> colonies holding the resulting constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, which is <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the general negative control. for background fluorescence. Measurement kits for P<sub>celA</sub> P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used as negative controls for function of &sigma;<sup>X</sup>.<br />
<br><br />
</p><br />
<p><br><b><u>Results</u></b> <br><br></p><br />
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<img src= "https://static.igem.org/mediawiki/2014/7/7f/PcelA%26comFA_macroscope.png" ><br />
<h5 style="font-size: 13px">Figure 1. P<sub>celA</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.</h5><br />
<h6 style= "font-size: 13px"> Only in the presence of &sigma;<sup>X</sup> would P<sub>celA</sub> and P<sub>comFA</sub> be turned on, as GFP expression could be seen when &sigma;<sup>X</sup> present. Therefore, &sigma;<sup>X</sup> is functional. P<sub>celA</sub> and P<sub>comFA</sub> gave little GFP signal in the absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter BBa_J23101 in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.</h6><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379000">P<sub>celA</sub> (BBa_K1379000)</a> and <a href= "http://parts.igem.org/Part:BBa_K1379001">P<sub>comFA</sub> (BBa_K1379001) </a></h3><br />
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<p class="first_letter_enhanced"> <br><br>For characterization, P<sub>celA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>celA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a>. <br><br><br />
<br />
P<sub>comFA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>comFA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a>. <br><br><br />
<br />
Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>celA</sub> and P<sub>comFA</sub> Measurement Kits with and without the &sigma;<sup>X</sup> generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the negative control for background fluorescence. <br><br><br />
<br />
Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). <i>E. coli</i> DH10B strains holding the constructs with or without &sigma;<sup>X</sup> generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> measurement device <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was measured alongside. The measurement was done with 3 replicas. <br />
</div><br><br><br />
<br />
<div class= "content_area_one_row"><br />
<div class="content_image"><br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src= "https://static.igem.org/mediawiki/2014/8/8c/Wiki_hkust_pcelA_pcomFA.png"/><br><br />
<h5 style="font-size: 13px">Figure 2. P<sub>celA</sub> has 0.53 RPU and P<sub>comFA</sub> hsa 1.21 RPU when paired with &sigma;<sup>X</sup> generator.</h5><br />
<h6 style= "font-size: 13px"> P<sub>celA</sub> and P<sub>comFA</sub> was measured in reference to <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> constitutive promoter with and without &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. RPU shown was calculated from 3 replicas.</h6> <br><br><br />
<br />
</div><br />
<br />
</div><br />
</td><br />
<br />
</tr><br />
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</table><br />
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<div class='content_1'><h3>Characterization Method</h3><br />
<table class="content_table" align= "center" ><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"> <br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct σ<sup>x</sup> Generator pSB3K3-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br />
<br />
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a> to pSB3k3.<br><br><br />
<br />
2. Construct σ<sup>x</sup> Generator pSB3K3-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> <br><br />
<br />
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a> to pSB3K3.<br><br><br />
<br />
3. Transforming pSB3K3-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
<br />
4. Transforming pSB3K3-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
5. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit<br><br><br />
<br />
6. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on procedure number 1. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values (we used Envision Multilabel Reader) every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
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<p><br><u><b>Equipments</b></u><br><br><br />
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</p><br />
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<br><br />
<u><b>References</b></u><br><br></p><br />
<p><br />
<br />
BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 and http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 <br />
<br><br><br />
<br />
Luo P., & Morrison D. (2003).<i> Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae</i>. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003<br />
<br><br><br />
<br />
Piotrowski A., Luo P., & Morrison D. (2009). <i>Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.</i> Journal of Bacteriology. doi:10.1128/JB.01750-08<br />
<br><br><br />
<br />
Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). <i>Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.</i> Molecular Systhetic Biology .doi:10.1038/msb.2013.58<br />
<br><br><br />
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J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterizationTeam:Hong Kong HKUST/pneumosensor/characterization2014-10-17T16:31:28Z<p>Jordyevan: </p>
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<div>{{Team:Hong_Kong_HKUST/shell|<br />
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<h2>Pneumosensor Characterization</h2><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379004"> σ<sup>x</sup>(BBa_K1379004) </a></h3><br />
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<p><br><b><u>Introduction</u></b> <br><br><br />
<p class="first_letter_enhanced">To test the functionality of &sigma;<sup>X</sup>, we first enable constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator, <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>.The generator was then assembled with the standard promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, with either promoter P<sub>celA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a>) and P<sub>comFA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a>). <i>E. coli</i> colonies holding the resulting constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, which is <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the general negative control. for background fluorescence. Measurement kits for P<sub>celA</sub> P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used as negative controls for function of &sigma;<sup>X</sup>.<br />
<br><br />
</p><br />
<p><br><b><u>Results</u></b> <br><br></p><br />
<div class="content_image"><br />
<img src= "https://static.igem.org/mediawiki/2014/7/7f/PcelA%26comFA_macroscope.png" ><br />
<h5 style="font-size: 13px">Figure 1. P<sub>celA</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.</h5><br />
<h6 style= "font-size: 13px"> Only in the presence of &sigma;<sup>X</sup> would P<sub>celA</sub> and P<sub>comFA</sub> be turned on, as GFP expression could be seen when &sigma;<sup>X</sup> present. Therefore, &sigma;<sup>X</sup> is functional. P<sub>celA</sub> and P<sub>comFA</sub> gave little GFP signal in the absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter BBa_J23101 in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.</h6><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379000">P<sub>celA</sub> (BBa_K1379000)</a> and <a href= "http://parts.igem.org/Part:BBa_K1379001">P<sub>comFA</sub> (BBa_K1379001) </a></h3><br />
<table class="content_table" align= "center" ><br />
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<br />
<p class="first_letter_enhanced"> <br><br>For characterization, P<sub>celA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>celA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a>. <br><br><br />
<br />
P<sub>comFA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>comFA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a>. <br><br><br />
<br />
Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>celA</sub> and P<sub>comFA</sub> Measurement Kits with and without the &sigma;<sup>X</sup> generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the negative control for background fluorescence. <br><br><br />
<br />
Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). <i>E. coli</i> DH10B strains holding the constructs with or without &sigma;<sup>X</sup> generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> measurement device <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was measured alongside. The measurement was done with 3 replicas. <br />
</div><br><br><br />
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<div class= "content_area_one_row"><br />
<div class="content_image"><br />
<img style="width:80%; display: block;<br />
margin-left: auto;<br />
margin-right: auto" src= "https://static.igem.org/mediawiki/2014/8/8c/Wiki_hkust_pcelA_pcomFA.png"/><br><br />
<h5 style="font-size: 13px">Figure 2. P<sub>celA</sub> has 0.53 RPU and P<sub>comFA</sub> hsa 1.21 RPU when paired with &sigma;<sup>X</sup> generator.</h5><br />
<h6 style= "font-size: 13px"> P<sub>celA</sub> and P<sub>comFA</sub> was measured in reference to <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> constitutive promoter with and without &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. RPU shown was calculated from 3 replicas.</h6> <br><br><br />
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<div class='content_1'><h3>Characterization Method</h3><br />
<table class="content_table" align= "center" ><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"> <br />
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<p><u><b>Construction</b></u><br><br><br />
1. Construct σ<sup>x</sup> Generator pSB3K3-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br />
<br />
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a> to pSB3k3.<br><br><br />
<br />
2. Construct σ<sup>x</sup> Generator pSB3K3-(<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> <br><br />
<br />
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a> to pSB3K3.<br><br><br />
<br />
3. Transforming pSB3K3-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
<br />
4. Transforming pSB3K3-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a><br><br><br />
<br />
5. Transforming pSB3K3-<a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit<br><br><br />
<br />
6. Transforming pSB3K3<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> (GFP generator) from the 2014 Distribution Kit.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on procedure number 1. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) with M9 minimal medium (we used Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile to culture the cells, and Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane.)<br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture (we used Multichannel Pipetman) and mix it with M9 medium in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom. (we used Micro test plate 96 well flat bottom, made by SARSTEDT.)<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned <i>E. coli</i> strains are cultured to mid-log phase (OD600 = 0.3 - 0.5) (we used Envision Multilabel Reader.)<br><br><br />
<br />
Filter used on Envision Multilabel Reader: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of pSB3K3-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
<br />
<p><br><u><b>Equipments</b></u><br><br><br />
<br />
<br />
</p><br />
</div><br />
</td><br />
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<p><br />
<br><br />
<u><b>References</b></u><br><br></p><br />
<p><br />
<br />
BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 and http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 <br />
<br><br><br />
<br />
Luo P., & Morrison D. (2003).<i> Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae</i>. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003<br />
<br><br><br />
<br />
Piotrowski A., Luo P., & Morrison D. (2009). <i>Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.</i> Journal of Bacteriology. doi:10.1128/JB.01750-08<br />
<br><br><br />
<br />
Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). <i>Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.</i> Molecular Systhetic Biology .doi:10.1038/msb.2013.58<br />
<br><br><br />
<br />
J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4<br />
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}}</div>Jordyevanhttp://2014.igem.org/Team:Hong_Kong_HKUST/pneumosensor/characterizationTeam:Hong Kong HKUST/pneumosensor/characterization2014-10-17T15:56:07Z<p>Jordyevan: </p>
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<h2>Pneumosensor Characterization</h2><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379004"> σ<sup>x</sup>(BBa_K1379004) </a></h3><br />
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<p><br><b><u>Introduction</u></b> <br><br><br />
<p class="first_letter_enhanced">To test the functionality of &sigma;<sup>X</sup>, we first enable constitutive expression of &sigma;<sup>X</sup> in the &sigma;<sup>X</sup> Generator, <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>.The generator was then assembled with the standard promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>, with either promoter P<sub>celA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379000">BBa_K1379000</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a>) and P<sub>comFA</sub> (Promoter only: <a href= "http://parts.igem.org/Part:BBa_K1379001">BBa_K1379001</a>, w/ <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>: <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a>). <i>E. coli</i> colonies holding the resulting constructs in pSB3K3 were observed under fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, which is <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the general negative control. for background fluorescence. Measurement kits for P<sub>celA</sub> P<sub>comFA</sub> without &sigma;<sup>X</sup> Generator were used as negative controls for function of &sigma;<sup>X</sup>.<br />
<br><br />
</p><br />
<p><br><b><u>Results</u></b> <br><br></p><br />
<div class="content_image"><br />
<img src= "https://static.igem.org/mediawiki/2014/7/7f/PcelA%26comFA_macroscope.png" ><br />
<h5 style="font-size: 13px">Figure 1. P<sub>celA</sub> and P<sub>comFA</sub> promoters activated in presence of &sigma;<sup>X</sup>.</h5><br />
<h6 style= "font-size: 13px"> Only in the presence of &sigma;<sup>X</sup> would P<sub>celA</sub> and P<sub>comFA</sub> be turned on, as GFP expression could be seen when &sigma;<sup>X</sup> present. Therefore, &sigma;<sup>X</sup> is functional. P<sub>celA</sub> and P<sub>comFA</sub> gave little GFP signal in the absence of &sigma;<sup>X</sup> but has comparable activity as reference promoter BBa_J23101 in presence of &sigma;<sup>X</sup>. Scale bar = 5mm.</h6><br />
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<div class='content_1'><h3> <a href= "http://parts.igem.org/Part:BBa_K1379000">P<sub>celA</sub> (BBa_K1379000)</a> and <a href= "http://parts.igem.org/Part:BBa_K1379001">P<sub>comFA</sub> (BBa_K1379001) </a></h3><br />
<table class="content_table" align= "center" ><br />
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<br />
<br />
<p class="first_letter_enhanced"> For characterization, P<sub>celA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>celA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379002">BBa_K1379002</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a>. <br><br><br />
<br />
P<sub>comFA</sub> promoter was assembled with the promoter measurement kit <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> to give the P<sub>comFA</sub> Measurement Kit <a href= "http://parts.igem.org/Part:BBa_K1379003">BBa_K1379003</a> in plasmid pSB3K3. The construct was further assembled with &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a> to give <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a>. <br><br><br />
<br />
Qualitative characterization was performed by comparing intensities of GFP signals from colonies of <i>E. coli</i> DH10B strain holding the P<sub>celA</sub> and P<sub>comFA</sub> Measurement Kits with and without the &sigma;<sup>X</sup> generator under a fluorescent macroscope with UV filter. Measurement kit for standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a>, <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> was used as a positive control; <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was used as the negative control for background fluorescence. <br><br><br />
<br />
Quantitative characterization was performed following the protocol described in “Measuring the activity of BioBrick promoters using an in vivo reference standard” (Kelly et al., 2009). <i>E. coli</i> DH10B strains holding the constructs with or without &sigma;<sup>X</sup> generator respectively were grown to mid-log phases. GFP intensities and cell densities were then sampled every 30 minutes for 5 consecutive time points to obtain growth rates and GFP synthesis rates. The GFP synthesis rates were then compared to that of standard reference promoter <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> measurement device <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> to obtain the Relative Promoter Units (RPUs). For subtraction of background fluorescence, pSB3K3 holding <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a> was measured alongside. The measurement was done with 3 replicas. <br />
<br />
</div><br />
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<div class= "content_area_one_row"><br />
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<img src= "https://static.igem.org/mediawiki/2014/8/8c/Wiki_hkust_pcelA_pcomFA.png"/><br><br />
<h5 style="font-size: 13px">Figure 2. P<sub>celA</sub> has 0.53 RPU and P<sub>comFA</sub> hsa 1.21 RPU when paired with &sigma;<sup>X</sup> generator.</h5><br />
<h6 style= "font-size: 13px"> P<sub>celA</sub> and P<sub>comFA</sub> was measured in reference to <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> constitutive promoter with and without &sigma;<sup>X</sup> generator <a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>. RPU shown was calculated from 3 replicas.</h6><br />
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<div class='content_1'><h3>Characterization Method</h3><br />
<table class="content_table" align= "center" ><br />
<tr class= "content_row"><br />
<td class= "content_cell"><br />
<div class= "content_area_one_row"> <br />
<br />
<p><u><b>Construction</b></u><br><br><br />
1. Construct σ<sup>x</sup> Generator (<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3<br><br />
<br />
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379005">BBa_K1379005</a> to pSB3k3.<br><br><br />
<br />
2. Construct σ<sup>x</sup> Generator (<a href= "http://parts.igem.org/Part:BBa_K1379006">BBa_K1379006</a>)-P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3 <br><br />
<br />
Or migrate <a href= "http://parts.igem.org/Part:BBa_K1379007">BBa_K1379007</a> to pSB3k3.<br><br><br />
<br />
3. Transforming P<sub>celA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3<br><br><br />
<br />
<br />
4. Transforming P<sub>comFA</sub>-<a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3<br><br><br />
<br />
5. Transforming <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a>-pSB3K3 (Standard Constitutive Promoter/Reference Promoter) from the 2014 Distribution Kit<br><br><br />
<br />
6. Transforming <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3 (GFP generator) from the 2014 Distribution Kit.<br><br><br />
<br><br />
<br />
<p><u><b>Measurement</b></u><br><br><br />
1. Preparing supplemented M9 medium <br>(M9 Minimal salt medium protocols could be seen on the <a href= "https://2014.igem.org/Team:Hong_Kong_HKUST/wetlab/protocols">Protocols</a> page, or download the <a href= "https://static.igem.org/mediawiki/2014/8/8b/M9_Minimal_medium_protocol.pdf">PDF</a> file) <br><br><br />
<br />
2. Culturing <i>E. coli</i> DH10B strain carrying the whole construct listed on procedure number 1. Grow cell culture overnight (Incubate 37°C and shake for 15 hours) in falcon tubes with M9 minimal medium; <br><br><br />
<br />
3. Take out 20-30μl of overnight cell culture with Multichannel Pipetman and mix it with M9 medium in the 96 Deep Well plate. <br><br><br />
<br />
4. Incubate in 37°C and shake for 3 - 4 hours.<br><br><br />
<br />
5. Take out 200ul of cells from the 96 deep well plates, and put it on a micro test plate 96 well flat bottom.<br><br><br />
<br />
6. Measuring the GFP intensity and OD595 values every 30 minutes after the above mentioned E. coli strains are cultured to mid-log phase (OD600 = 0.3 - 0.5);<br><br><br />
<br />
Filter used: <br><br />
- Absorbance :Photometric 595nm,<br><br />
- Excitation :485nm FITC,<br> <br />
- Emission :535nm FITC, <br><br />
- Mirror module : FITC (403) at bottom. <br><br><br />
<br />
- In between measurements, keep incubating the cells in 37°C while shaking. <br><br><br />
<br />
7. Calculating the Relative Promoter Units (RPU) using the obtained data; <br><br><br />
</p><br />
<br />
<br />
<p><br><u><b>Data Processing</b></u><br><br><br />
1. After <i>E. coli</i> carrying the right construct was grown to mid-log phase, GFP intensity and OD595 were measured every 30 minutes (up to 120min); <br><br><br />
<br />
2. GFP intensity are subtracted with the background fluorescence which is the fluorescence of <a href= "http://parts.igem.org/Part:BBa_E0240">BBa_E0240</a>-pSB3K3. Curve reflecting GFP expression change was plotted (from 4 measurements from time=0 to time=120); OD595 was converted to OD600, and average values were taken; <br><br><br />
<br />
<br />
3. GFP synthesis rate was then obtained by calculating the slope of the above mentioned curve; <br><br><br />
<br />
4. Absolute promoter activity of P<sub>celA</sub>, P<sub>comFA</sub>, and <a href= "http://parts.igem.org/Part:BBa_I20260">BBa_I20260</a> were calculated by dividing the GFP synthesis rate with the average OD600 value; <br><br><br />
<br />
5. Averaged absolute promoter activity was then obtained by averaging the respective 3 sets of absolute promoter activity values; <br><br><br />
<br />
<br />
6. Finally, R.P.U was calculated by dividing the averaged P<sub>celA</sub> and P<sub>comFA</sub> absolute promoter activity over the averaged <a href= "http://parts.igem.org/Part:BBa_J23101">BBa_J23101</a> absolute promoter activity. R.P.U value of P<sub>celA</sub> and P<sub>comFA</sub> reflect the maximum GFP expression in the presence of σ<sup>x</sup>. Leakage could be analyzed according to the R.P.U value that shows the GFP expression of P<sub>celA</sub> and P<sub>comFA</sub> promoter in the absence of σ<sup>x</sup>.<br><br><br />
Equation of the RPU calculation is shown below: <br><br />
<br />
<img style= "width:50%" src= "https://static.igem.org/mediawiki/2014/e/e0/RPUequation_ust2014.png"/><br />
<br><br><br />
<br />
<p><br><u><b>Equipments</b></u><br><br><br />
1. Multichannel Pipetman.<br><br />
2. Corning® 96 well storage system storage block, 2 mL, V-bottom, sterile. <br><br />
3. Corning® microplate sealing tape white Rayon (with acrylic), sterile, suitable for cell/tissue culture applications, breathable sterile membrane. <br><br />
4. Micro test plate 96 well flat bottom, made by SARSTEDT. <br><br />
5. Envision Multilabel Reader.<br />
<br />
<br />
</p><br />
</div><br />
</td><br />
<br />
<br />
<br />
<br />
<!-- end of one row of content , two column one picture left--><br />
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<table class="content_table" align= "center" ><br />
<tr class= "content_row" ><br />
<td class= "content_cell" colspan= "2"><br />
<div class= "content_area_two_row"><br />
<p><br />
<br><br />
<u><b>References</b></u><br><br></p><br />
<p><br />
<br />
BioCyc was retrieved from http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 and http://www.biocyc.org/SPNE171101/NEW-IMAGE?type=GENE&object=GJC8-867 <br />
<br><br><br />
<br />
Luo P., & Morrison D. (2003).<i> Transient Association of an Alternative Sigma Factor, ComX, with RNA Polymerase during the Period of Competence for Genetic Transformation in Streptococcus pneumoniae</i>. Journal of Bacteriology. doi:10.1128/JB.185.1.349-358.2003<br />
<br><br><br />
<br />
Piotrowski A., Luo P., & Morrison D. (2009). <i>Competence for genetic transformation in Streptococcus pneumoniae: termination of activity of the alternative sigma factor ComX is independent of proteolysis of ComX and ComW.</i> Journal of Bacteriology. doi:10.1128/JB.01750-08<br />
<br><br><br />
<br />
Rhodius V., Segall-Shapiro T., Sharon B., Ghodasara A., Orlova E., Tabakh H., . . . Voigt C. (2013). <i>Design of orthogonal genetic switches based on a crosstalk map of σs, anti-σs, and promoters.</i> Molecular Systhetic Biology .doi:10.1038/msb.2013.58<br />
<br><br><br />
<br />
J. R. Kelly, A. J. Rubin, J. H. Davis, J. Cumbers, M. J. Czar, ..., D. Endy. (2009). Measuring the activity of BioBrick promoters using an in vivo reference standard. <i>Journal of Biological Engineering</i>, 3, 4. doi: 10.1186/1754-1611-3-4<br />
<br />
<br />
<br />
<br />
<br />
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}}</div>Jordyevanhttp://2014.igem.org/File:Wiki_hkust_pcelA_pcomFA.pngFile:Wiki hkust pcelA pcomFA.png2014-10-17T15:55:18Z<p>Jordyevan: </p>
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