Team:TUDelft/ResultsTest

Sci-Phi 29

Overview

Parts Construction

text


Part Characterization

text



Orthogonalibity

text


Orthogonal Replication

text

Toxicity Assay

text


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:

Transcriptional variation

Translational variation

Expression across different organisms

Cross species codon harmonization


Future Plan

text



References