Genome engineering is an important technological advance. In particular, genome engineering of bacterial genomes such as E coli should be quick and simple.
This is the vector being created in order to test the hypothesis of whether or not 100bp overlaps can modify the genome when provided from a plasmid.
This would be important because then only ~250bp of synthesis (about 25-50$) will be able to make a point mutation in the E coli genome in a rapid and easily multiplexed way. In order to get the highest efficiency possibly, I am putting the CRISPR guide RNAs under the rhamnose promoter. Once the population is homogenized with my plasmid, I will cut and select for survivors of the CRISPR. By offsetting the cut stage, it will make these cells easier to transform and work with, as you can simply infect with viruses which have packaged this plasmid. I will update this page once I get results using the above plasmid for genomic modifications.
See multigene CRISPR below for more information on the CRISPR system I will be using.