Team:William and Mary/Parts
All Parts (By Category) In deciding which parts to submit to the iGEM Registry we focused on three main aspects. 1. Ensuring our project is as reproducible and extensible as possible. To that end we have submitted all of new composite fluorescent protein parts that we constructed during the project. 2. Making genome integration as straightforward as possible for iGEM teams. In order to accomplish this goal we designed, tested, and validated a new integrator cassette that allows for simple genome integration using either 3A or Gibson Assembly. 3. Increasing the number of tools available for promoter-mediated regulation in synthetic biology. We created and validated an E. coli codon optimized dCas9 variant and a suite of gRNAs to target the most commonly used promoters in iGEM. Integrator Cassette For our method of genome integration (notebook / protocol link) the input is linear DNA, generated by PCR, containing what you would like to integrate onto the genome and an antibiotic resistance cassette to allow for selection. The galK Integrator allows digestion with the standard BioBrick enzymes and 3A assembly of your part of interest to create the integration construct (see left).
This product can then be amplified using primers (details found here) and then used in the integration protocol. We have successfully used the integrator to incorporate stretches of DNA up to 2.1kb into the galK locus, not including the resistance cassette (1179 bp).
