Team:William and Mary/Composite Part
Composite Parts Of our 25 total parts, 24 were composite parts.
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. (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.Gilbert, Luke A., et al. "Genome-scale CRISPR-mediated control of gene repression and activation." Cell 159.3 (2014): 647-661. 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.)
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 (http://parts.igem.org/Part:BBa_K1795001) 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 (link to John’s part about this) and further analyzed. References UPDATE THESE: 1: Elowitz, Michael B., et al. "Stochastic gene expression in a single cell." Science 297.5584 (2002): 1183-1186. 2: Raser, Jonathan M., and Erin K. O'Shea. "Noise in gene expression: origins, consequences, and control." Science 309.5743 (2005): 2010-2013.
