Team:BostonU/Repressors

From 2014.igem.org

Repressor Proteins and Promoters

Why Repressor Proteins?

In order to build and test the priority encoder below, we will need to create new basic parts for new repressors and their corresponding promoters. The diagram below shows an Abstract Genetic Regulatory Network, or AGRN, where only the function is outlined without any specific parts assigned to the promoters, repressors, or small molecules (with the exception of the fluorescent proteins GFP, RFP, and BFP).

Our current MoClo library of parts does not contain enough orthogonal repressors and promoters to flush this design out. Based on this limitation, we began a literature search to find five new repressor and promoter pairs that could work together in one system. Early on in our search we found the 2014 article by Stanton et al. [1] where they built and tested a library of 73-TetR family repressors from various prokaryotic genomes. From these parts, they showed that 20 of them had strong transfer curves when turned on (Figure 4 below). From that subset, we chose five repressors that had desirable but slightly different transfer curves from one another: LmrA, SrpR, PhlF, BetI, and BM3R1. For our definition of desirable, we wanted to see a clear on and off function across at least two decades of REU with varying peak fluorescence as measured by Stanton et al..

Figure 4 from Stanton et al.(2014) where each repressor (noted in bottom left of each graph) was under control of an IPTG-inducible promoter and the corresponding repressor promoter controlled YFP fluorescence.

Design and Assembly

After choosing the five repressors and their promoters, we filled in the AGRN with specific parts as a first pass to start the design process. Without any assignments of parts, there are over 5,000 variants possible. As a first pass, we decided to assign our parts naively (see below) just to get started on designing oligos to clone the parts. We chose pTetR, pBad, and pLacI as the three small molecule promoters because we already have those in our library of parts.

We have obtained plasmid DNA from AddGene for LmrA, SrpR, PhlF, BetI, and BM3R1 and have designed oligos to clone these as MoClo CDS parts with the C fusion site on the 5' side and the D fusion site on the 3' side to fit into our current library format. It should be noted that we are not generating fusion proteins with GFP until we finish testing our current fusion proteins.

Testing

We are still in the process of cloning these new parts. Once the parts are built, we plan to test them using the design schematic shown below. We will use an inducible promoter and build a transcriptional unit (TU) with the repressor protein and then build a second TU with the matching repressible promoter controlling GFP expression. We will measure GFP using flow cytometry and, depending on which inducible promoter we select (pBad, pLacI, or pTetR), we will induce the system with the corresponding small molecule (arabinose, IPTG, or aTc, respectively).

This is our planned testing device for the repressors (rep) and their respective promoters (pRep).

References

[1] B. Stanton, A. Nielsen, A. Tamsir, K. Clancy, T. Peterson & C. Voigt (2014). "Genomic mining of prokaryotic repressors for orthogonal logic gates." Nature Chemical Biology 10: 99-105. doi:10.1038/nchembio.1411

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