Team:William and Mary/Parts

NOISE - W&M iGEM

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

BBa_K1795023


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).

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Antibiotic Operon

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dCas9s

BBa_K17950001

BBa_K17950012


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.

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gRNAs

Part:BBa_K179500021

Part:BBa_K179500032

Part:BBa_K179500043

Part:BBa_K179500054

Part:BBa_K179500065

Part:BBa_K179500076

Part:BBa_K179500087

Part:BBa_K179500098

Part:BBa_K179500109

Part:BBa_K1795001110

Part:BBa_K1795001210


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.

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XFPs Under Various Promoters

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G^2

Part:BBa_K17950221


This plasmid (http://parts.igem.org/Part:BBa_K1795022) 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 (http://parts.igem.org/Part:BBa_K1795023).

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