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). Antibiotic Operon Detail1 Detail2 Detail3 Detail4 text text dCas9s Part BBa_K1795000 is a dCas9 protein-coding region that has been optimized for expression in E. coli. Additionally, we created a functional dCas9 operon (http://parts.igem.org/Part:BBa_K1795001) that, when transformed into E. coli is constitutively expressed. For further details, please see our Basic Part page. gRNAs To complete our creation of a codon-optimized dCas9 parts, we created functional gRNAs that target the most commonly used promoters in iGEM. These parts (BBa_K1795002- BBa_K1795012), when transformed into E. coli, constitutively express gRNA that, in complex with our dCas9 variant, will repress transcription of the targeted promoter. We have also contributed a scramble gRNA (Part:BBa_K17950013) that does not target any region in the E. coli genome and can be used as a negative control. For further details about our gRNAs, please see our Part Collections page.
CFPs/YFF Under Various Promoters Detail1 Detail2 Detail3 Detail4 In order to test multiple promoters for their intrinsic noise, we needed to construct composite parts that had the structure seen to the right.
In each construct the RBS and DT were kept constant throughout, but the promoter used to drive transcription was changed. Each construct was made in a CFP and YFP variant. Both the CFP and YFP used in this project have an LVA tag, decreasing the half-life of the protein. All of these parts have been sequenced confirmed and functionally validated. text Dual Fluorescent Plasmid This plasmid consists of both a CFP-LVA driven by R0010 and YFP-LVA driven by R0010. This allows future teams to use this part to investigate transcriptional noise either on a low copy number plasmid or by integrating this part using our galK integrator.
