Team:William and Mary/Basic Part
Basic Part Despite the extensive characterization of the average strength of the promoters available on the Biobrick registry, very few have information pertaining to the variability in their expression. This variability, also commonly referred to as stochasticity or noise, in gene expression can be split into two components: extrinsic and intrinsic noise.
CRISPR/Cas9 has seen an explosion in use over the last five years, both within and outside of iGEM. Recent developments have extended the functionality of the Cas9 protein by modifying the nuclease domain to remove its catalytic function. This Cas9 variant, dCas9, can be used to simply target and bind to DNA. If dCas9 is targeted to a promoter, this binding causes steric hindrance, preventing RNAP from binding to and activating transcription [1-3].
However, despite several iGEM teams using both Cas9 and dCas9 variants in previous years, there have been no codon-optimized versions of the Cas9 protein made for E. coli. 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 that, when transformed into E. coli is constitutively expressed.
(RIGHT) This part has been functionally validated by showing 97% repression of R0010-driven expression of RFP when co-transformed with BBa_K1795002. Once individual cell measurements have been taken for both CFP and YFP expression using confocal microscopy, the intrinsic noise measurements can be calculated and further analyzed. References: 1: Bikard, David, et al. "Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system." Nucleic acids research41.15 (2013): 7429-7437. 2: Gilbert, Luke A., et al. "Genome-scale CRISPR-mediated control of gene repression and activation." Cell 159.3 (2014): 647-661. 3: Qi, Lei S., et al. "Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression." Cell 152.5 (2013): 1173-1183. 