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Team:UCLA/Project/ProjectDesign
Project Design
Inspired by the natural Bordetella phage diversity-generating retroelement (DGR) system, we sought to develop an in vitro analog that could produce even greater numbers of possible target-binding variants.
To this end, we first isolated the major tropism determining (mtd) gene from the Bordetella phage genome, which was generously donated to us by Jeff Miller’s lab at UCLA. Initial attempts at moving the components of the DGR into E. coli has not successfully yielded a functional product. However, the Mtd protein is still a choice candidate for our project given its ability to resist structural deformation following mutagenesis of the variable binding region. Without the native reverse transcriptase-mediated homologous recombination system to generate diversity, we designed a series of overlapping oligomers with mutagenic islands that bind to the mtd gene’s variable region and introduce random nucleotides during PCR. The farthest oligomer at the 3’ end of the gene also contains a FLAG-tag and a Serine-Glycine linker that would facilitate purification from undesirable byproducts during mRNA display.
<Insert the mutagenic primer sequences; perhaps with an annotation below the figure explaining how the ‘N’s in the sequence allow random incorporation of nucleotides during PCR?>
This in vitro system has several advantages over the natural DGR, and boasts even greater possible genetic diversity. Naturally, only adenine bases within the mtd gene’s variable region are modifiable. While this still provides a significantly large number of possible variants, the primers used in our experiments do not follow the same adenine site-specific mutation restrictions, and permit entire regions to be modified. Primer-mediated mutagenesis thus provides even greater sequence variation than the normal DGR system.
The number of variants will invariably decrease in the steps following library generation. Ideally, the library should contain several copies of each possible variant before beginning mRNA display, so that sufficient diversity is retained for the screen.
While shallow sequencing coverage does not allow for accurate calculations of copies per mtd variant within the library, the maximum amount of diversity within a given sample can be calculated based on the concentration of DNA.
<Maybe a calculator that people can use for copy number? Otherwise, we can include the sample calculation for maximum diversity in a defined sample>
