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.
Two-Plasmid System localizes fluorescent proteins
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.
Fluorescent microscopy demonstrates increased localization of fluorescent proteins when the EutS plasmid is present. We have demonstrated and characterized this using three different EutS promoter systems and found that of the three, EutS Mid and Low (BBa_2129004,005) produce the best results. Some characterization was attempted using an IPTG-inducible cassette, however due to the medium no particular difference in expression was observed in non-IPTG media.
Localization of the EutC1-19 - eGFP system was tested with low, high, and arabinose cassettes on pSB1K3. Localization was always noticeable with good levels of EutS expression, however, cells would frequently overexpress eGFP resulting in sporadic cells being extremely fluorescent and making visualization of surrounding cells more difficult. The best results can be seen with EutS with a low cassette (BBa_2129006) and araC-Pbad (BBa_K808000)under non-arabinose added medium using Luria-Bertani broth (this is documented behavior on the parts page for BBa_K808000).