As mentioned in the discussion of our InterLab results, there has been success in the transduction of Mycobacterium cells using mycobacteriophages. These protocols require growing up cells, exposing cells to bacteriophages & conducting a plaque assay. The preliminary results from our study show that fluorescence can be detected from as early as 20-60 minutes of E. coli growth. This would greatly reduce the time frame of pathogen detection. The results of the InterLab study also show that the protocol is adaptable to accommodate different laboratory environments.
BioBricks For Mutagenesis
The short tail fibre (stf) gene was successfully reconstructed with ORF-401 & ORF-314. No mutagenesis work was carried out on stf. However, the BioBrick is now available for mutagenesis work & future testing for changes in binding specificity.
The tail fibre assembly (tfa) protein acts as a molecular chaperone to stf. The cloning of tfa will permit future mutagenesis work, which may facilitate the assembly of mutant stf if problems arise when these mutations are introduced. tfa was only cloned into a circuit & not made into a standalone BioBrick, after which this construct was cloned into a circuit where it is repressed by TetR. The addition of anhydrous tetracycline will alleviate the repression and therefore allow optimisation of tfa expression to give the controlled production of recombinant virions.
BioBricks for the Lytic/Lysogenic Cycle Control
For the biosynthesis of bacteriophage virions for our product, it is essential that the lytic & lysogenic cycles are tightly regulated in order to maximise the number of virions produced. The cI & Cro genes were successfully constructed into a circuit. With cI being constitutively expressed, lysogeny can be maintained for bacterial load to be increased in order to maximise the number of bacteriophage virions to be produced. Meanwhile, the production of T7 RNAP is controlled under a pBAD (araC-regulated) promoter which in turn stimulates the expression of Cro. This control can allow optimisation of when to stimulate the switch from lysogeny to lytic cycle to maximise viral titre in the lysate.
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