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.

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. We compared our wild-type T7 promoter based iFFL system to a iFFL system based on a T7 promoter variant (Ryo Komura et al., 2018) with 50% strength compared to the wild-type (medium T7 based iFFL system). Constructs used: [insert images]

  Results

  The robustness of the iFFL circuits to transcriptional rates variation was validated by measuring GFP fluorescence using flow cytometry. During initial verification of the iFFL system, unrepressed iFFL was used as a positive control. Unexpectedly, low fluorescence was observed for this control. From flow cytometry results, it was seen that the cells containing the unrepressed iFFL system were smaller in size and had different cell morphology (region R1) than E.coli cells without any plasmid (region R0) and therefore can not be compared to each other. The difference in cell morphology suggests that over expression of TALE and GFP could be burdensome to the cells. Hence, for further data analysis, GFP expressed from T7sp1 was used as a control. For the flow cytometry measurements, E.coli BL21 DE (3) without plasmid were used to correct for background fluorescence. Only cells of similar size and complexity (forward scatter and side scatter) were compared, by gating the most dense region of E. coli BL21DE(3). All measurements were done in logarithmic growth phase under 1mM IPTG induction and the median fluorescence of the gated populations were compared (figure ()). Figure () shows similar GFP fluorescence despite change in promoter strengths. This demonstrates gene expression independent of transcriptional rates.

• Results -- Independence to IPTG concentration﹀

  To validate our model prediction, we designed T7 promoters based iFFL systems. We compared our wild-type T7 promoter based iFFL system to a iFFL system based on a T7 promoter variant (Ryo Komura et al., 2018) with 50% strength compared to the wild-type (medium T7 based iFFL system). Constructs used: [insert images]

  Results

  The robustness of the iFFL circuits to transcriptional rates variation was validated by measuring GFP fluorescence using flow cytometry. During initial verification of the iFFL system, unrepressed iFFL was used as a positive control. Unexpectedly, low fluorescence was observed for this control. From flow cytometry results, it was seen that the cells containing the unrepressed iFFL system were smaller in size and had different cell morphology (region R1) than E.coli cells without any plasmid (region R0) and therefore can not be compared to each other. The difference in cell morphology suggests that over expression of TALE and GFP could be burdensome to the cells. Hence, for further data analysis, GFP expressed from T7sp1 was used as a control. For the flow cytometry measurements, E.coli BL21 DE (3) without plasmid were used to correct for background fluorescence. Only cells of similar size and complexity (forward scatter and side scatter) were compared, by gating the most dense region of E. coli BL21DE(3). All measurements were done in logarithmic growth phase under 1mM IPTG induction and the median fluorescence of the gated populations were compared (figure ()). Figure () shows similar GFP fluorescence despite change in promoter strengths. This demonstrates gene expression independent of transcriptional rates.

Translational variation

• Design﹀

Expression across different organisms

• Design﹀

Cross species codon harmonization

• Design﹀

Future Plan

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