<figcaption align="center">Two-Plasmid System localizes fluorescent proteins</figcaption>
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We used standard BioBrick plasmids pSB4A5 and and introduced high (BBa_314100), low (BBa_314100), and lactose (BBa_K314103) promoters with the EutS compartmental structure protein (BBa_2129001), along with a EutC1-19 tagged enhanced GFP fluorescent protein (BBa_2129003) on pSB1K3 with the same high and low promoters as well as an arabinose inducible promoter (BBa_2129006, 007). Comparative localization was demonstrated by comparison to fluorescent microscopy of non-EutS expressing cells. </p>
We used standard BioBrick plasmids pSB4A5 and and introduced high (BBa_314100), low (BBa_314100), and lactose (BBa_K314103) promoters with the EutS compartmental structure protein (BBa_2129001), along with a EutC1-19 tagged enhanced GFP fluorescent protein (BBa_2129003) on pSB1K3 with the same high and low promoters as well as an arabinose inducible promoter (BBa_2129006, 007). Comparative localization was demonstrated by comparison to fluorescent microscopy of non-EutS expressing cells. </p>
We set out at the beginning of the summer with the aim of consistently producing a bacterial microcompartment from a single protein, with the objective of adding a non-canonical amino acid at a point within its sequence that enables us to break these self-forming compartments apart using light. We have produced the following results:
1. We have successfully produced a one-protein BioBrick compatible part that forms microcompartments (EutS, BBa_K2129001)
2. We have tested and demonstrated the results of different expression levels of our EutS compartment with variable levels of tagged eGFP using a variable two-plasmid system and a variety of promoters (BBa_K2129003-K2129007)
3. We successfully mutated amber stop codons at at least three loci in the EutS
4. We introduced a 3-plasmid system using the AzoPhe pEVOL plasmid from the Shultz lab ( shultzlablink) and then visualized the results of adding irradiated phenylalanine-4’-azobenzene to the medium of cells with the mutated plasmids
5. We attempted to produce similar results using genome modification of NEB-5alpha e.coli cells, and produced a library of genomic edits towards this end.
Construction of the EutS BioBrick compatible part
Using the part EutSMNLK (BBa_K311004), we PCR amplified out the S protein of the operon and cloned it alone onto a plasmid backbone. Testing using our two-plasmid system has demonstrated that EutS does indeed produce localization of EutC tagged fluorescent proteins, as described in Schmidt-Dannert et al. 2016 (PMID: 27063436). Our system is fully BioBrick compatible and extremely simple, producing localization and compartmentalization with a minimum of necessary protein expression.