Our team's mission was to set the basis for a universal mobile genetic tool which could be readily used to engineer a wide range of diverse bacterial species, contributing to a more streamlined, host-independent multi-chassis Synthetic Biology. To achieve this, we set ourselves two main goals: our first goal was to investigate the implementation and viability of the phi29 bacteriophage linear DNA replication system as an orthogonal replication tool in bacterial cells. Our second goal was to engineer a system which generates constant gene expression levels independently of transcriptional and translational variations that are expected to occur when transferring genetic circuits across organisms.
In this project, we have successfully demonstrated independence of expression levels from transcriptional variations with our novel incoherent feed-forward loop (iFFL) design, and have achieved cross-species expression level stabilization between E. coli and P. putida. Moreover, we have validated replication of our synthetic phi29 plasmid in cell-free systems as well as performed titration experiments to uncover the effects of different concentrations of phi29 replication-associated proteins in E. coli.
Cell-free Replication of Linear DNA Construct
We demonstrated succesful in vitro replication of our own linear construct (oriL-GFP-kan-oriR). The protocol can be found here.
Figure 1: (Left) Design of in vitro translation and transcription reaction to amplify our own linear construct (oriL-GFP-Kan-oriR) in vitro. A plasmid expressing DNAP and TP (van Nies et al., 2018) and purified p5 and p6 were added to the reaction for in vitro replication of the construct. In vitro transcription and translation of the TP-DNAP plasmid results in DNAP and TP proteins. These proteins together with p5 and p6 replicate the linear construct in the PUREfrex system. (Right) Purified OriL-GFP-Kan-OriR from a coupled DNA transcription-translation and replication reaction with (+) and without (-) dNTPs, amplified in vitro using the PUREfrex 2.0 system. An intense band of the expected size (~2.3 kb) is observed from the +dNTPs reaction, demonstrating the successful OriL-GFP-Kan-OriR in vitro replication.
Successful replication of our linear construct has been shown on the agarose gel (Figure 1). A very intense band is visible at a size of 2.3 kb, corresponding to the size of OriL-GFP-Kan-OriR construct. This indicates that there has been successful in vitro amplification of our construct.
Figure 2: Design of our orthogonal transcription iFFL for the stabilization of expression across organisms. T7 promoter variants ensure constant ratio of transcription in varying T7 RNAP concentrations and activity. The use of the same RBS in both repressor and output gene result constant ratio of translation initiation in both genes. This results in independence of gene expression levels from variations in these rates.
Based on kinetic model solutions, we predicted that the system would be stable in GFP expression independently of variations in transcription. Firstly, we demonstrated this control across varying T7 RNAP concentrations (caused by different IPTG titrations in the E. coli BL21 expression strain).
Figure 3: Flow cytometry media GFP fluorescence of iFFL optimized construct (red square) and unregulated GFP under T7sp1 promoter (blue circle) in E. coli BL21 DE3 at different IPTG concentrations.
When compared to uncontrolled GFP, the iFFL clearly exhibited steady expression in the range of 0 to 1 mM IPTG concentrations.
We also showed that promoter strength did not have an effect on the control of gene expression by an iFFL. We tested this by comparing promoters of different strengths under control of the iFFL to promoters of similar strength without any repression.
Figure 4: Flow cytometry median GFP fluorescence of WT T7 promoter iFFL system, unregulated version of this construct (no operator present), medium T7 promoter iFFL system, and optimized iFFL system with weak promoter driving TALEsp1.
As Figure 4 shows, both the native strength and medium strength T7 promoter versions of our iFFL exhibited similar fluorescence. Furthermore, tunability was shown by the change in promoter strength ratios, in the case of a weak promoter in TALEsp1 repressor. The data corroborates with the effective repression of our novel TALE regulated T7 promoters. For more details click here.
Cross-species Gene Expression Stabilization
We have succeeded in cloning a broad host range promoter version of our iFFL (Figure 5) in P. putida for comparison with E. coli, as well as negative control constitutive promoter.
Figure 5: Design of our broad host range transcription iFFL for the stabilization of expression across organisms. The system was tested in E. coli and P. putida.
In Figure 6 we observed a reduction in both absolute and relative differences of gene expression levels. Although these constructs are influenced by multiple variables, the data suggests that our approach is a right step for the gene level stabilization across organisms. Calibration of the system for higher expression of gene of interest would further validate the control of expression. In conclusion, we have successfully engineered an iFFL which functions effectively in both E. coli and P. putida.