The expression levels in a genetic circuit are strongly correlated to the plasmid copy number of the DNA template Segall-Shapiro et al., (2018) The amount of gene plasmid copy number can change when the plasmid is transferred between organisms. Therefore there is a need for expression levels independent of plasmid copy number if the same genetic circuit is used in different organisms. The steady-state solution of our model tells us that when our repressor binding is fully non-cooperative, we have complete independence of plasmid copy number.

The model without assumptions has the same expression level independent of plasmid copy number (figure ). We therefore can transfer our circuit between organisms and expect the expression of the gene of interest to be independent of the changes in plasmid copy number of our orthogonal plasmid.

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 demonstrate gene expression independent of copy number driven by Universal Bacterial Expression Resource (UBER) system, we first cloned our T7 promoter based optimized iFFL into low and medium copy number backbones ( pICH82113 , and pICH82094 respectively) from the MoClo toolkit. The clones were then co-transformed with the UBER portable T7 expression system. The UBER system expresses T7 RNAP at a stable level as described on our design page . The host cells for co-transformation were E.coli Top 10 which are not engineered to express T7 RNAP.

</p>

                               <img src="" style="width:80%;border:1px solid #00a6d6;" class="centermodel"
                                    alt="TALE system">
                               <figcaption class="centermodel"> Figure 1: Fluorescence histogram of co-transformations obtained by flow cytometry measurements during the logarithmic growth phase.The curves show raw fluorescence values of E. coli Top10 cells without a plasmid (black), clones co-transformed with T7 promoter based optimized iFFL in low copy number backbone and UBER  (green), clones co-transformed with T7 promoter based optimized  iFFL system in medium copy number backbone and UBER (pink).   
</figcaption> 
 

From figure 1, a clear shift in fluorescence for clones co-transformed with UBER system from E.coli Top 10 cells is observed. This demonstrates that the UBER system successfully drives the iFFL system. Since, the overlap in the fluorescence curves is large gene expression independent of copy number was not demonstrated. Therefore, we believe further optimization is required to develop a system combining the portable T7 expression system and iFFL.

((Raw data from flow cytometry experiments can be found on T7 promoter based optimized iFFL </ul> </li> </ul>

Conclusion

               </div>

                   Behavior of promoters (transcriptional rates) significantly changes across different bacterial hosts <a href=”https://www.ncbi.nlm.nih.gov/pubmed/29061047”> (Yang S et al., 2017) </a>. 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 <a href="https://2019.igem.org/Team:TUDelft/Model#TranscriptionalVariations" > modeling </a>. 
                   
 

• <a class="toggle " href="javascript:void(0);" >Results -- Independence to IPTG concentration﹀</a>

  Aside from testing gene expression independent of transcriptional variation by using promoters of different strengths, the effect of different concentrations of IPTG on the iFFL loop was tested. Change in IPTG concentrations, changes in-vivo concentrations of T7 RNAP and this contributes to variations in transcriptional rates. In unrepressed systems, the expression of the GOI is a function of IPTG concentrations. However, in iFFL systems, since the transcriptional rates of TALE and GFP are under control of T7 promoters, similar GOI expression is expected (figure 1). As a control we expressed GFP under the control of T7sp1 promoter was used (figure 2). <img src="" style="width:60%;border:1px solid #00a6d6;" class="centermodel" alt="TALE system"> <figcaption class="centermodel">Figure 1: Optimized T7 based iFFL. TALE is under cotntrol of weak T7 promtoer. GFP is controlled by a T7 promoter. </figcaption>
  <img src="https://static.igem.org/mediawiki/2019/4/4e/T--TUDelft--notale.png#00a6d6;" class="centermodel" alt="TALE system"> <figcaption class="centermodel">Figure 2: Negative control, T7 promoter controlling GFP. </figcaption>

  The output GFP fluorescence was measured using flow cytometry during logarithmic growth phase after induction with 1mM IPTG. 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

  <p> Figure 3 the GFP fluorescence of the unrepressed control changes with changing concentrations of IPTG while the iFFL system shows the same GFP expression across different IPTG concentrations. Thus, the iFFL system has been shown to insulate gene expression against changes in transcription rates (achieved by varying IPTG concentrations). .

                             <img src="https://static.igem.org/mediawiki/2019/a/a9/T--TUDelft--IPTGtitration.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
                                  alt="TALE system">
                             <figcaption class="centermodel">Figure 3: 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>
                             
  
  

• <a class="toggle " href="javascript:void(0);" >Results -- Tunability﹀</a>

  The predictions made by modeling not only tell us that we can maintain the same level of gene expression but also that we can easily tune the expression levels by changing one of the promoters. By changing one of the promoters to another variant of T7 we can expect a different level of expression, while at the same time expect it to behave similarly when transferred between organisms. Through the use of T7 variants we could achieve wide ranges of expression levels, which can be used to establish complex genetic circuits, while also expecting it to work similarly in different biological contexts.
  
  We tested the prediction by changing the promoter controlling the TALE protein to a variant of T7 which has been shown to be about 10% in strength compared to the wild-type (Ryo Komura et al., 2018). According to the model the expression should increase.

  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

  <p> Figure 1 clearly shows higher fluorescence when a lower T7 promoter version is used to express the TALE protein in comparison to our systems with the same ratio in promoter strengths for both genes.

                             <img src="https://static.igem.org/mediawiki/2019/5/5d/T--TUDelft--tunability.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
                                  alt="TALE system">
                             <figcaption class="centermodel">Figure 1: Steady-state GFP fluorescence measurement of E. coli BL21DE(3) cells expressing our iFFL systems. The graph depicts a different T7 iFFL systems, one with both promoters T7, one with both medium strength and one where the promoter controlling of the TALE is weak. As a control GFP under control of an unrepressed T7 promoter was used. </figcaption>
                             
  
  

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.

                   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). 

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

To achieve similar gene expression across different organisms the iFFL system needs to be robust to changes in copy number, transcriptional and translational rates. We experimentally demonstrated gene expression independent of transcriptional variations and through modelling showed adaptation variations in copy number and translational variation of our iFFL system. Through the implementation of the iFFL loop using engineered broad host range promoters, we successfully demonstrated similar GFP expression across E. coli and P. putida. Thereby demonstrating gene expression insulated from variations associated to microbial hosts.

• <a class="toggle " href="javascript:void(0);" >Results -- Expression across organisms﹀</a>

  Broad host range promoter (P_BHR) was designed by combining the conserved -10 and -35 regions from E.coli and B.subtilis <a href=""> (Yang S et al., 2018) </a> and the promoter was engineered to contain a binding site for TALE repressor (P_BHRsp1). Using the P_BHR and P_BHRsp1, we constructed an iFFL genetic circuit driving GFP expression. The circuit was transformed in E. coli and P. putida and, output fluorescence was measured by flow cytometry during logarithmic growth phase. To correct for background fluorescence, E. coli and P. putida without plasmids were used as blanks. GFP under the control of P_BHRsp1 was used as a positive (unrepressed) control. As the cell morphologies of E. coli and P. putida are different they cannot be compared directly, gating was based on the most dense regions in the scatter plot for each organism. In order to compare the GFP expression levels between each organism, the background fluorescence for each organism was subtracted by its respective blank .

  Results

  <p> Figure 1 clearly shows higher fluorescence when a lower T7 promoter version is used to express the TALE protein in comparison to our systems with the same ratio in promoter strengths for both genes.

                             <img src="https://static.igem.org/mediawiki/2019/4/4a/T--TUDelft--difforgs.svg" style="width:60%;border:1px solid #00a6d6;" class="centermodel"
                                  alt="TALE system">
                             <figcaption class="centermodel">Figure 1: : Steady-state GOI production while translation rates of both TALE and GOI are changed. The lines indicate the constant rate of the translation rates. </figcaption>
                             
  
  

  In figure 1, the median fluorescence of the gated populations is plotted. Significantly large difference in expression levels is observed between the unrepressed controls and the broad host range promoter based iFFL systems in E.coli and P.putida. However, similar levels of expression were observed from iFFL systems in E.coli and P.putida . The difference in expression levels between the unrepressed circuit is significantly higher than the difference in expression levels between the iFFL system (578530 and 2351.2 respectively).

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

• <a class="toggle " href="javascript:void(0);" >Design﹀</a>

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References

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