Team:Hong Kong HKUST/riboregulator/characterization

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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  
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  
activate the PBAD promoter.</p>
activate the PBAD promoter.</p>
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<div class='content_1'><h3>PBAD promoter BBa_I0500 in Part Registry</h3>
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<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>
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<div class='content_1'><h3>Results</h3>
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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 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>
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<h5 style="font-size: 13px">Figure 3. RPU of PBAD promoter in three different cell strains across different arabinose concentration. </h5>
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<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>
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<p><b><u>RPU of PBAD in different cell strains</u></b><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 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>
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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>
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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 PBAD¬ in DH5α.<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>
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<p><b><u>P<sub>BAD</sub> Leakage</u></b><br>
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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>
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<img style="width:80%; display: block;
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    margin-right: auto" src="http://2014.igem.org/wiki/images/5/54/Pbad_RPU_2.png" />
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<h5 style="font-size: 13px">Figure 4. Leakage of Pbad promoter in different cell strain. </h5>
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<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>
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<p><b><u>3-D graphs for DH10B and DH5α</u></b><br>
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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>
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    margin-right: auto" src="http://2014.igem.org/wiki/images/3/33/3dgraph_pbad.png" />
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<h5 style="font-size: 13px">Figure 5.  </h5>
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<div class='content_1'><h3>Discussion</h3>
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<p>Groningen 2011 results may not truly represent the gradual induction of PBAD promoter <br><br>
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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>
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<div class='content_1'><h3>Methods</h3>
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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>
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<img style= "width:50%" src= "http://2014.igem.org/wiki/images/6/60/Riboregulator_ust_Equation.png"/> <br><br>
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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.
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Revision as of 20:33, 17 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.
Table 1 List of riboregulator pairs characterized by HKUST iGEM 2014 team:

Name and registry code Group Cognate pair
Lock 1 (BBa_J01010) Key 1 (BBa_J01008) iGEM 2005_Berkeley (Golden Bear) Yes
Lock 3 (BBa_J01080) Key3 (BBa_J01086) iGEM 2005_Berkeley (Golden Bear) Yes
Medium lock (BBa_K175031)Key for medium lock (BBa_K175032) iGEM09_TUDelft Yes
Lock 1 (BBa_J01010) Key 1 (BBa_J01008) iGEM 2005_Berkeley (Golden Bear) Yes
Lock 1 (BBa_J01010) Key 1 (BBa_J01008) iGEM 2005_Berkeley (Golden Bear) Yes
Lock 1 (BBa_J01010) Key 1 (BBa_J01008) iGEM 2005_Berkeley (Golden Bear) Yes

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


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. This is 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 (source) (Figure 1. A). (fsd different between –TA and -CR).



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). 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). For HKUST lock 1 and HKUST key 1 cognate pair, some repression is observed, but the system 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 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, 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 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. After repression, the system needs to be activated when taRNA is expressed.




PBAD Characterization


Introduction

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. 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 PBAD promoter transcribes the araC gene in the opposite direction. AraC protein is responsible to repress the activity 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 activate the PBAD promoter.


PBAD promoter BBa_I0500 in Part Registry

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


Results

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).


Figure 3. RPU of PBAD promoter in three different cell strains across different arabinose concentration.
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.


RPU of PBAD in different cell strains
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).

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.

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.

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.

PBAD Leakage
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.

Figure 4. Leakage of Pbad promoter in different cell strain.
For each strain, fluorescence at no arabinose induction was normalized to the fluorescence at maximum observed fluorescence. The column graphs represent triplicate mean ± SD.


3-D graphs for DH10B and DH5α
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.)

Figure 5.


Discussion

Groningen 2011 results may not truly represent the gradual induction of PBAD promoter

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.


Methods

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.



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.



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