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Team:Edinburgh/project/cisgenic
From 2014.igem.org
Cisgenic wires
Yuma Shino
This (Cis-genic Metabolic Wiring) was intended to be constructed from genes only found in strains of E. coli. Similar to how it was done by Rafael Silva-Rocha and Victor de Lorenzo, where they used genes found in Pseudomonas putida strains, we aimed to create a signalling system using genes were responsive to aromatics in our host organism which were strains of E. coli.
Contrary to our first expectations metabolic pathways which were regulated by different substrate specific promoters were not as prevalent as claimed and it took a while to find one that satisfied the following requirements:
- It was controlled by a substrate specific promoter.
- The end product of the pathway was the substrate of another pathway, which also was controlled by a substrate specific promoter.
- The product was either the co-activator or inducer of the other pathway.
- The product of the pathway was freely diffusible.
Three pathways were, eventually, chosen to create this system. One pathway would act as a sender and two others were selected for the receiving strain in order to allow for possible failures of one of the other selected pathways. For this the aromatic amine degradation pathway was selected as a sender pathway. The two receiver pathways were the phenylacetate (PA) catabolic pathway and the 4-hydroxyphenylacetic (HPA) acid catabolic pathway.
The system was intended to signal via the transfer of either phenylacetic acid (PA) or 4-hydroxyphenylacetic acid (4HPA) between E.coli cells that had the aromatic amine degradation pathway and one that had the phenylacetate catabolic pathway or the 1-hydroxyphenylacetic acid catabolic pathway.
The substrates required for this were tyramine and phenylethylamine (PEA) which were converted into 4HPA and PA respectively by the aromatic amine degradation pathway.
The genes we used were:
- FeaR
- PaaX
- HpaR
The above are transcription factors.
FeaR is an activator that binds with an unknown substrate of the aromatic amine degradation pathway. The entire aromatic amine degradation pathway can be activated with the substrate PEA or tyramine.
PaaX is a repressor of the paa cluster and induced off with PA.
HpaR is one of the repressor of the hpa cluster which encodes the genes required for HPA degradation. It could be induced off with 4HPA.
The promoters we planned to use were:
- PtynA (Finely Tuned Regulation of the Aromatic Amine Degradation Pathway in Escherichia coli)
- PpaaA
- PhpaG (Molecular determinants of the hpa regulatory system of Escherichia coli: the HpaR repressor)
As the products of the aromatic amine degradation pathway are often metabolised into the TCA cycle eventually, the idea of removing downstream pathways in the sender strain to increase the amount of signal produced was proposed. This was also to prevent self-signalling in receiver strains as well, by removing the upstream pathways so they could not create the signal they were supposed to receive by themselves.
The relevant plasmids can be seen here:
