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.

  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.

  We therefore designed our genetic circuit such that the transcriptional units of TALE and GFP are oriented in opposite directions.

• Results -- Independence to IPTG concentration﹀

  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. The ability of the construct to insulate gene expression from transcriptional rate changes was validated by induction of clones (host E.coli BL21 DE(3) cells) with different IPTG concentrations. 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. We tested this by inducing our T7 promoter based optimized iFFL .. with different levels of IPTG: 0, 0.1, 0.5 and 1 mM. As a control, GFP under the control of T7sp1 .. was used 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.
  (see figure 1).

  Results

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.

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

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Future Plan

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