Difference between revisions of "Team:TUDelft/ResultsTest"

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                                 <h3>Results</h3>
 
                                 <h3>Results</h3>
 
                                 <p>Unexpectedly low fluorescence was observed for our control 1. However, when comparing the unrepressed iFFL system forward and side-scatter to <i>E. coli</i> BL21DE(3) cells without any plasmid they were smaller in size and had . The difference in cell morphology suggests that over expression of TALE and GFP could be burdensome to the cells. Hence, for further data analysis, only control 2 was used.</p> <br>
 
                                 <p>Unexpectedly low fluorescence was observed for our control 1. However, when comparing the unrepressed iFFL system forward and side-scatter to <i>E. coli</i> BL21DE(3) cells without any plasmid they were smaller in size and had . The difference in cell morphology suggests that over expression of TALE and GFP could be burdensome to the cells. Hence, for further data analysis, only control 2 was used.</p> <br>
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                                    <img src="https://static.igem.org/mediawiki/2019/5/5c/T--TUDelft--promoter_variation_wetlab_model.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
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                                <img src="https://static.igem.org/mediawiki/2019/5/5c/T--TUDelft--promoter_variation_wetlab_model.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
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                                        alt="TALE system">
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                                    alt="TALE system">
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                                    <figcaption class="centermodel"><b>Figure 6</b>: Steady-state GFP fluorescence measurement of promoter variation using flow cytometry. The graph depicts T7 and medium T7 iFFL systems, expected to give the same fluorescence according to the model. As a control, GFP under control of an unrepressed T7 promoter was used. </figcaption>
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                                <figcaption class="centermodel"><b>Figure 6</b>: Steady-state GFP fluorescence measurement of promoter variation using flow cytometry. The graph depicts T7 and medium T7 iFFL systems, expected to give the same fluorescence according to the model. As a control, GFP under control of an unrepressed T7 promoter was used. </figcaption>
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                                    <br>
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                                <br>
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                                    <p>In figure ..., we can clearly see that the stabilized systems are very close in fluorescence while the unrepressed system is higher, as predicted.</p>
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                                <p>In figure ..., we can clearly see that the stabilized systems are very close in fluorescence while the unrepressed system is higher, as predicted.</p>
 
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                         <a class="toggle " href="javascript:void(0);" ><b>Results -- Independence to IPTG concentration</b><span style="float:right;"><b>&#xfe40;</b></span></a>
 
                         <a class="toggle " href="javascript:void(0);" ><b>Results -- Independence to IPTG concentration</b><span style="float:right;"><b>&#xfe40;</b></span></a>
 
                         <ul class="inner accordion">
 
                         <ul class="inner accordion">
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                             <p> Apart from demonstrating gene expression independent of promoter strengths, for the system to be versatile, different levels of gene expression needs to be demonstrated. To attain higher level of GFP expression, we expressed TALE with a lower strength T7 promoter compared to the promoter expressing GFP.
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                             <p>  
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                                The ability of the construct to insulate gene expression from transcriptional rate changes was validated by induction of clones (host <i> E.coli </i> BL21 DE(3) cells) with different IPTG concentrations.  
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                                Aside from transcriptional variation through changing the promoters in the construct, we also tested the effect of different concentrations of IPTG. In unrepressed systems the expression of the GOI increases when more IPTG concentrations are increased.  
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                                 Different levels of IPTG were used to obtain different transcriptional rates as the in-vivo concentration of T7 RNAP is a function of concentration of IPTG. Therefore, GFP expression would increase with increasing IPTG concentrations.  However, in our iFFL system the transcription rate of the TALE protein will increase in the same ratio and thus we can expect the same level of expression.
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                                 Since both the promoter of the TALE and the promoter of the GFP are variants of T7 they will both increase in the same way and should result in the same level of expression. As a control we expressed GFP under the control of our <a href="http://parts.igem.org/Part:BBa_K2918010">T7sp1 promoter</a>.                            
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                                We tested this by inducing our <a href="http://parts.igem.org/Part:BBa_K2918010" ><b> T7 promoter based optimized iFFL </b>.</a>. with different levels of IPTG: 0, 0.1, 0.5 and 1 mM. As a control, GFP under the control of <a href="http://parts.igem.org/Part:BBa_K2918010" ><b> T7sp1 </b>.</a>. was used
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                                In figure (), it is observed that GFP fluorescence of the control increases with IPTG concentrations while the T7 based optimized iFFL showed similar gene expression despite changes in IPTG concentrations.    <br>
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                                (see figure 1).
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                             </p>
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                            <p>We measure GFP fluorescence using flow cytometry. As a reference for background fluorescence we use <i>E. coli</i> BL21DE(3) cells without any plasmid. The most dense region (determined by eye) in the scatter plot (forward and side-scatter) is selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram is gated to discern between cells that are "off" or "on" (expressing GFP or not), by gating the <i>E. coli</i> BL21DE(3) cells without any plasmid</p>. <br>
 +
                            <br>
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                            <p>The median of the background is subtracted from the samples and are compared. </p>
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                             <h3>Results</h3>
 
                             <h3>Results</h3>
 
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                             <p>
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                             </p>
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                             <p> Figure ... clearly shows a strong increase in fluorescence when no repression is used. However, when our iFFL is implemented the levels of GFP are constant.
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                            <img src="https://static.igem.org/mediawiki/2019/a/a9/T--TUDelft--IPTGtitration.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
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                                alt="TALE system">
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                            <figcaption class="centermodel"><b>Figure ...</b>: Steady-state GFP fluorescence measurement of IPTG titration using FACS. The graph depicts a T7 iFFL system induced using different levels of IPTG, which according to the model should give the same result. As a control GFP under control of an unrepressed T7 promoter was used. </figcaption>
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                            <br>
 
                         </ul>
 
                         </ul>
 
                     </li>
 
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Revision as of 00:07, 21 October 2019

Sci-Phi 29

Overview

Parts Construction

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Part Characterization

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Orthogonalibity

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Orthogonal Replication

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Toxicity Assay

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Controllabillity

Overview

The behavior of genetic parts and circuits in different bacterial species is unpredictable as it is influenced by host-dependent variations. We identified the variables to be: copy number, transcriptional and translational rates. We implemented a unique control system motif (incoherent feed forward loop) into a genetic circuit to achieve gene expression independent of copy number, transcriptional and translational rates.

Construction

We modeled the genetic implementation of the iFFL loop and varied the identified variables. Based on the results from the modeling, we made design choices.

  • Results﹀
      We learned through the implementation of the model that constant transcriptional and translational rates of TALE and GFP needs to be maintained to achieve gene expression independent of transcriptional and translational variations respectively. TALE system
      Figure 5: Steady-state GFP production while transcription rates of both TALE and GOI are changed (aT/aG = constant). The lines indicate constant ratio of transcription rates

      To achieve constant ratios of transcriptional rates of TALE and GFP, we used the orthogonal T7 promoter and its variants to express TALE and GFP genes. The following constructs were successfully cloned by Golden Gate Assembly. To achieve constant ratios of translational rates for TALE and GFP, we used the same ribosome binding sites for the expression of TALE and GFP. Furthermore, to demonstrate expression independent of translational rates, we switched constructed circuits with different RBSs. When transcriptional units are placed in series, leaky expression of the gene in the second transcriptional unit can occur. This is due to the efficiency of the terminator of the first transcriptional unit. The model shows that leaky expression significantly affects the ability of the iFFL system to adapt to changes in copy number.

      TALE system
      Figure 9: Comparison of a perfect terminator and a leaky terminator on the expression level at different plasmid copy number.
      We therefore designed our genetic circuit such that the transcriptional units of TALE and GFP are oriented in opposite directions.

Copy number independence

Copy number of plasmids vary when used in different bacterial hosts and this significantly alters behaviour of parts. To achieve higher predictability of parts across different bacterial species, we aimed to demonstrate independence to copy number of our iFFL systems, as predicted by our modeling . Furthermore, to facilitate the development of portable gene expression systems and reduce host dependency, the iFFL system was successfully expressed along with the Universal Bacterial Expression Resource (UBER) system (Kushwaha & Salis, 2015).
  • Experimental design﹀

      To test the independence to copy number we cloned our T7 promoter based optimized iFFL and a control into low and medium copy number backbones ( pICH82113 , and pICH82094 respectively) from the MoClo toolkit.



      To reduce dependency on host transcriptional machinery, we co-transformed these constructs with the UBER portable T7 expression system. The UBER system expresses T7 RNAP at a stable level as described on our design page . Constructs used:

      Results

      We ....

Transcriptional variation

Behavior of promoters (transcriptional rates) significantly changes across different bacterial hosts (Yang S et al., 2017) . Hence, promoters either need to be re-characterized for each bacterial hosts or promoters specific to the host need to be identified. Using iFFL, we demonstrated gene expression independent of transcriptional rates when the transcription rate of both genes (TALE and GOI) maintain the same ratio, as predicted by our modeling .

  • Results -- Independence to promoter strengths﹀

      To validate our model prediction, we designed T7 promoters based iFFL systems with varying promoter strengths. We compared our wild-type T7 promoter based iFFL system to a iFFL system based on a T7 promoter variant with 50% strength compared to the wild-type (Ryo Komura et al., 2018) (medium T7 based iFFL system). Two negative controls were used:

      1. Control 1: T7 based iFFL system without a binding site for TALE protein in the promoter controlling GFP.
      2. Control 2: A promoter controlling GFP without any TALE expressed.

      In figure ..., an overview of all the constructs is depicted.

      [insert images]

      We measure GFP fluorescence using flow cytometry. As a reference for background fluorescence we use E. coli BL21DE(3) cells without any plasmid. The most dense region (determined by eye) in the scatter plot (forward and side-scatter) is selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram is gated to discern between cells that are "off" or "on" (expressing GFP or not), by gating the E. coli BL21DE(3) cells without any plasmid

      .

      The median of the background is subtracted from the samples and are compared.

      Results

      Unexpectedly low fluorescence was observed for our control 1. However, when comparing the unrepressed iFFL system forward and side-scatter to E. coli BL21DE(3) cells without any plasmid they were smaller in size and had . The difference in cell morphology suggests that over expression of TALE and GFP could be burdensome to the cells. Hence, for further data analysis, only control 2 was used.


      TALE system
      Figure 6: Steady-state GFP fluorescence measurement of promoter variation using flow cytometry. The graph depicts T7 and medium T7 iFFL systems, expected to give the same fluorescence according to the model. As a control, GFP under control of an unrepressed T7 promoter was used.

      In figure ..., we can clearly see that the stabilized systems are very close in fluorescence while the unrepressed system is higher, as predicted.

  • Results -- Independence to IPTG concentration﹀

      Aside from transcriptional variation through changing the promoters in the construct, we also tested the effect of different concentrations of IPTG. In unrepressed systems the expression of the GOI increases when more IPTG concentrations are increased. Since both the promoter of the TALE and the promoter of the GFP are variants of T7 they will both increase in the same way and should result in the same level of expression. As a control we expressed GFP under the control of our T7sp1 promoter.

      We measure GFP fluorescence using flow cytometry. As a reference for background fluorescence we use E. coli BL21DE(3) cells without any plasmid. The most dense region (determined by eye) in the scatter plot (forward and side-scatter) is selected for gating in order to only compare cells of similar morphology. Furthermore, the fluorescence histogram is gated to discern between cells that are "off" or "on" (expressing GFP or not), by gating the E. coli BL21DE(3) cells without any plasmid

      .

      The median of the background is subtracted from the samples and are compared.

      Results

      Figure ... clearly shows a strong increase in fluorescence when no repression is used. However, when our iFFL is implemented the levels of GFP are constant. TALE system

      Figure ...: Steady-state GFP fluorescence measurement of IPTG titration using FACS. The graph depicts a T7 iFFL system induced using different levels of IPTG, which according to the model should give the same result. As a control GFP under control of an unrepressed T7 promoter was used.

    • Conclusion

    Results above indicate successful implementation of the iFFL system to insulate from transcriptional variations. Transcriptional variations were achieved by using T7 promoters of different strengths and by induction at different IPTG concentrations. Furthermore, we demonstrated the tunability of the iFFL system to achieve different levels of gene expression. As predicted by our model, we achieved gene expression independent of changes in transcriptional rates by maintaining constant ratios of transcriptional rates of TALE and GFP genes.

Translational variation

In order to see the effect of translational variation on the expression levels of the gene of interest (GOI) we modeled our system for a range of translational rates for both genes (TALE and GOI).
  • Results﹀

      As can be seen in figure … the steady-state expression levels of GFP remain the same when the translation rates are kept constant.

Conclusion

According to our model solution we can maintain the same level of GOI expression when both translation rates remain constant. We therefore designed our system to contain the same RBS in front of both TALE and the GOI.

Expression across different organisms

We have demonstrated transcriptional variation independence and showed through modeling how the iFFL system is completely independent to copy number. Furthermore, we designed our construct with the same RBS based on modeling results. We combined the independence to all of these variables (copy number, transcriptional and translational variations) by testing our system in two different organisms (P. putida and E. coli).
  • Results -- Expression across organisms﹀

      Design: We made a broad host range promoter (Pbhr) version of our iFFL. The Pbhr iFFL system contains a promoter which has been shown to work in a wide range of organisms (Yang S et al., 2017) . We transformed both P. putida and E. coli with our Pbhr iFFL, and as a control GFP under the control of the same promoter was used. Fluorescence was measured by flow cytometry. For both organisms, cells without the plasmid were used to discern between background fluorescence. Gating was based on the most dense region for each organism, only cells of similar size and complexity were compared. P. putida and E. coli can’t be compared directly as they have different cell morphologies and background fluorescence. In order to compare the GFP expression levels in each organism we subtracted the background fluorescence for each organism respectively.

      In figure … we clearly see a very large difference in expression levels when the expression of GFP is not repressed by the TALE protein. However, the difference in expression is 2 orders of magnitude lower when the iFFL system is used.

Conclusion

The implementation of the iFFL significantly decreased the differences in expression levels between organisms. Our system sets the basis for controllability across organisms.

Cross species codon harmonization


Future Plan

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References