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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). </p> | 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). </p> | ||
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<figcaption align="center">Two-Plasmid System localizes fluorescent proteins</figcaption> | <figcaption align="center">Two-Plasmid System localizes fluorescent proteins</figcaption> | ||
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<h3> Mutation of EutS Sequence to introduce Amber stop codons </h3> | <h3> Mutation of EutS Sequence to introduce Amber stop codons </h3> | ||
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− | Using site directed mutagenesis, we changed the sequence of our EutS Low plasmid construct to instead use a Mid promoter, and we introduced amber stop codons into the sequence of EutS at residues that we selected based on predictive protein modelling with Rosetta and Pymol. Sequencing confirmed that several of these plasmids were successfully mutated at the loci we selected, including the part EutS | + | Using site directed mutagenesis, we changed the sequence of our EutS Low plasmid construct to instead use a Mid promoter, and we introduced amber stop codons into the sequence of EutS at residues that we selected based on predictive protein modelling with Rosetta and Pymol. Sequencing confirmed that several of these plasmids were successfully mutated at the loci we selected, including the part EutS Mutant C (BBa_2129002) which we characterized more fully with our three plasmid system and fluorescent microscopy. </p> |
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− | <figcaption align="center"> | + | <figcaption align="center">Amber EutS Mutant C</figcaption> |
− | <h3> Introduction of | + | <h3> Introduction of azo-phenylalanine amino acids to EutS structure </h3> |
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Using QuikChange PCR mutagenesis, we introduced Amber (UAG) stop codons to the EutS sequence at residues predictively modelled to produce super and substructure altering effects. Once sequenced and confirmed for incorporation of amber codons at desired sites, we performed a co-transformation of eGFP Low (BBa_2129006) and the pEVOL AzoPhe plasmid provided by the Shultz lab, and then introduced our mutated EutS Mid plasmids via electroporation. | Using QuikChange PCR mutagenesis, we introduced Amber (UAG) stop codons to the EutS sequence at residues predictively modelled to produce super and substructure altering effects. Once sequenced and confirmed for incorporation of amber codons at desired sites, we performed a co-transformation of eGFP Low (BBa_2129006) and the pEVOL AzoPhe plasmid provided by the Shultz lab, and then introduced our mutated EutS Mid plasmids via electroporation. | ||
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<figcaption align="center">3 plasmid system</figcaption> | <figcaption align="center">3 plasmid system</figcaption> | ||
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<figcaption align="center">EutS Mutants with and without AzoPhe</figcaption> | <figcaption align="center">EutS Mutants with and without AzoPhe</figcaption> | ||
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Latest revision as of 23:03, 3 November 2016
Results
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 gene
4. We introduced a 3-plasmid system using the AzoPhe pEVOL plasmid 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.