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Team:Technion/Project/RNAPs
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Overview
The purpose of different RNAPs used in our project is to control expression from suitable promoters inserted into phage genome. Addition of inducers to the medium will trigger transcription of polymerases needed to promote transcription of phage’s genes, thus allowing the phage to complete its lytic cycle. At first we thought to use three native polymerases to control our AND gates – T7, T3 and SP6 RNAPs. Those polymerases arise from T7, T3 and SP6 bacteriophages, respectively and belong to the same family of polymerases. They were chosen to be used in our project for 2 main reasons {1}:
- Those polymerases have high specificity recognizing their own promoters, but not native E.coli‘s or other polymerases’ promoters, those minimizing dangers of unwanted cross-reactivity.
- pT7, pSP6 and pT3 are very strong promoters, so their activity is very tightly regulated by inducers that can't be found in a specific E.coli we used in our project. Also, using this promoters prevents possible leakiness.
However, we had discovered that phage polymerases can exhibit toxicity to host cells, so we began to search after less toxic variants. Luckily, Ilya have found an article published by Chris Voight et al. Chris’ research focused around creating a set of orthogonal polymerases based on T7 wild type (WT) RNA polymerase (RNAP) that will be less toxic to the host cells and will recognize different promoters.
Chris had agreed to share his work with our team, so in total during our project we had worked with 6 different polymerases: T7 and SP6 WT RNAPS and eT7, eK1F, eN4, eT3 mutants produced by Chris and his team (‘e’ before RNAP’s name means engineered polymerase).
