DESIGN OVERVIEW

In our project we try to repurpose a peptide communication machinery from a phage of the Gram-positive Bacillus subtilis to the cells of Gram-negative Escherichia coli. To do so, we had to cross the inter-species barrier which proved to be a challenging task.

First, in the original system, the ‘Arbitrium’ peptide is secreted with a Bacillus secretion tag and processed into the mature peptide SAIRGA by a Bacillus enzyme [1]. To produce SAIRGA in E. coli, we had to replace this tag by an E. coli secretion signal. So, we have designed genes for three versions of SAIRGA containing different secretion signals: OmpA, PelB and Tat. To be considered as efficient, these secretion tags have to be cleaved after the secretion of the peptide. More details in The Peptide page.

Then, we had to design the reporter gene. The idea was to put the GFP under the control of pAimX promoter and to constitutively express AimR, which is the transcription factor. Thus, the GFP expression is controlled by an AimR regulated promoter, which activity is inhibited by the SAIRGA peptide. To be able to see quick changes in the GFP expression, we added a LVA degradation tag to the GFP, and we chose to use the sfGFP, which is more resistant to the addition of tags. More details in The Promoter page.

Beside, we wanted to switch our system and transform the activatable promoter in Bacillus to a repressible promoter in E. coli (see why on the Integrated Human Practices page). So, we created another construct using the original promoter as an operator sequence downstream of a constitutive E. coli promoter. Consequently, we developed a promoter which responds to a constitutively expressed repressor and a “de-repressor”, the peptide. More details in The Promoter page.

In this project, we have codon optimized all our CDS for E. coli chassis using JCat, used constitutive promoters to control the peptides and AimR genes, and designed strong RBSs for every CDS using Salis’s RBS Calculator [2, 3].

Finally, in order to perform a perfect activation and a perfect repression of the GFP production, we have used three different plasmids to clone our genes. We wanted to ensure that, in our system, the peptide was more concentrated than the receptor and the receptor was more concentrated than the promoter (in terms of copy number). So, we cloned our peptides in pSB1C3, a high copy plasmid, we cloned AimR in pSB3T5, a medium copy plasmid, and we cloned the pAimX promoter(s) and the sfGFP in pSB4K5, a low copy plasmid. In addition, the modelling team has realised that AimR is potentially toxic for the cell, which is one additional argument not to use it on a high copy plasmid.

REFERENCES

[1] Erez Z, Steinberger-Levy I, Shamir M, Doron S, Stokar-Avihail A, Peleg Y, Melamed S, Leavitt A, Savidor A, Albeck S, Amitai G, Sorek R. Communication between viruses guides lysis-lysogeny decisions. Nature (2017) 541, 488-493.

[2] Espah Borujeni A, Channarasappa AS, Salis HM. Translation rate is controlled by coupled trade-offs between site accessibility, selective RNA unfolding and sliding at upstream standby sites. Nucleic Acids Res (2014) 42, 2646-2659.

[3] Salis HM, Mirsky EA, Voigt CA. Automated design of synthetic ribosome binding sites to control protein expression. Nat Biotechnol (2009) 27, 946-50.