Team:Hong Kong HKUST/riboregulator/characterization
From 2014.igem.org
Line 86: | Line 86: | ||
<td class= "content_cell"> | <td class= "content_cell"> | ||
<div class= "content_area_one_row"> | <div class= "content_area_one_row"> | ||
- | <a href="#nogo"></a><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br><img style="width: | + | <a href="#nogo"></a><img src="https://static.igem.org/mediawiki/2014/f/fe/HKUST_ribo_characterizationA.png" /><br><img style="width:90%;" src="https://static.igem.org/mediawiki/2014/c/cc/HKUST_ribo_characterizationB.png"/> |
<h5 style="font-size: 13px">Figure 1. Fluorescence (F)/OD600 measurements of riboregulator pairs after arabinose induction and their corresponding controls. </h5> | <h5 style="font-size: 13px">Figure 1. Fluorescence (F)/OD600 measurements of riboregulator pairs after arabinose induction and their corresponding controls. </h5> | ||
Line 219: | Line 219: | ||
<br> | <br> | ||
- | <img style="width: | + | <img style="width:80%; display: block; |
margin-left: auto; | margin-left: auto; | ||
margin-right: auto" src="https://static.igem.org/mediawiki/2014/0/02/FACS_hkust.png" /> | margin-right: auto" src="https://static.igem.org/mediawiki/2014/0/02/FACS_hkust.png" /> | ||
- | <h5 style="font-size: 13px">Figure | + | <h5 style="font-size: 13px">Figure 2. Forward scatter intensity (FSC) versus GFP graphs for samples with P<sub>BAD</sub> promoter regulating GFP generator. </h5> |
<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> | <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> | ||
Line 233: | Line 233: | ||
</p> | </p> | ||
- | <img style="width: | + | <img style="width:50%; display: block; |
margin-left: auto; | margin-left: auto; | ||
margin-right: auto" src="https://static.igem.org/mediawiki/2014/8/88/Percentageofcells_pbad_HKUST.png" /> | margin-right: auto" src="https://static.igem.org/mediawiki/2014/8/88/Percentageofcells_pbad_HKUST.png" /> | ||
- | <h5 style="font-size: 13px">Figure | + | <h5 style="font-size: 13px">Figure 3. The percentage of cells in induced and uninduced state, and RPU across different arabinose concentration. </h5> |
<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> | <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> | ||
Line 247: | Line 247: | ||
margin-left: auto; | margin-left: auto; | ||
margin-right: auto" src="https://static.igem.org/mediawiki/2014/a/ab/Pbad_RPU_3strains.png" /> | margin-right: auto" src="https://static.igem.org/mediawiki/2014/a/ab/Pbad_RPU_3strains.png" /> | ||
- | <h5 style="font-size: 13px">Figure | + | <h5 style="font-size: 13px">Figure 4. RPU of P<sub>BAD</sub> promoter in three different cell strains across different arabinose concentration. </h5> |
<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> | <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> | ||
Line 268: | Line 268: | ||
margin-left: auto; | margin-left: auto; | ||
margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /> | margin-right: auto" src="https://static.igem.org/mediawiki/2014/3/33/3dgraph_pbad.png" /> | ||
- | <h5 style="font-size: 13px">Figure | + | <h5 style="font-size: 13px">Figure 5. Fluorescence and OD600 measurements of DH10B and DH5α induced in different arabinose concentrations. </h5> |
<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> | <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> | ||
</p> | </p> |
Revision as of 02:02, 18 October 2014
Riboregulator Characterization
Introduction
Riboregulator is a type regulatory RNA that can regulate translation. One component of the riboregulator system,
cis-repressing RNA (crRNA). CrRNA contains a cis-repressing sequence which is located 5’ of the RBS and the gene of interest.
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
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
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
team characterized 4 riboregulator already available in the Part Registry and 1 riboregulator introduced by our team.
Riboregulators have cognate pairs. For certain crRNA, there is a corresponding taRNA that can activate “unlock” the repression by crRNA. We originally thought that 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 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 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. |
Riboregulator Results
Figure 1. Fluorescence (F)/OD600 measurements of riboregulator pairs after arabinose induction and their corresponding controls.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 (BBa_J01010) and Key 1 (BBa_J01008) cognate pair. (C) Measurement for Lock 3(BBa_J01080) and Key 3 (BBa_J01086) cognate pair. (D) Measurement for Medium lock (BBa_K175031) and Key for medium lock (BBa_K175032) cognate pair. (E) Measurement for Lock 3c (BBa_J23031) and Key 3c (BBa_J23008).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 (BBa_J23102 ) 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 PBAD promoter (BBa_I0500 ). 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). 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 BBa_B0034 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 (BBa_B0034 ). 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. 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. |
Discussion
The fold increase for Lock 1- Key 1 is lower than that of riboregulator pair mentioned in the Isaacs et al.’s paper |
Methods
Fluorometry |