Difference between revisions of "Team:William and Mary/Composite Part"

 
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                                                 <p class="h2WM">Composite Parts</p>
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                                                 <p class="h2WM">Composite Part</p>
 
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                                                 <p>Of our 25 total parts, 24 were composite parts.
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                                                 <p>Our submission for Best Composite Part is the <b>galK Integrator</b>.
 
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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.)
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<a href=""></a>Driven by our insights about the <a href="https://2015.igem.org/Team:William_and_Mary/Modeling">increase in transcriptional noise strength from plasmid copy fluctuations</a>, we decided to provide future teams with a simple way to better manage the noise in the expression of a key protein in their genetic networks by restricting the gene copy fluctuations to within 1 and 2 copies. The galK Integrator is a part which allows easy integration of any sequence onto the galK locus of the E. coli chromosome, which was the locus used by Elowitz <i>et al.</i> to successfully integrate a functional cyan fluorescent protein with an antibiotic resistance cassette [1].
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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.  
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<div><p><div><p style="float: right;"><IMG SRC="https://static.igem.org/mediawiki/2015/6/6f/WMdcasIm.jpeg" width=600px></p><p>(RIGHT) This part has been functionally validated by showing 97% repression of <a href = "http://parts.igem.org/Part:BBa_R0010">R0010</a>-driven expression of RFP when co-transformed with BBa_K1795002.</p></div></p>
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<a href="https://2015.igem.org/Team:William_and_Mary/Protocols:Integration">For our method of genome integration</a> 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 below). This product can then be amplified using primers (details found here) and then used in the integration protocol. We have successfully used this part to integrate a 2.1kb segment attached to a 1.1kb antibiotic resistance cassette.  
  
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<div><p>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.</p></div>
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<div><p style="float: right;"><IMG SRC="https://static.igem.org/mediawiki/2015/b/ba/WMIntegratorDiagram.png" width=600px></p><p>(RIGHT) The galK Integrator will serve to promote the reproducibility of our noise measurements, encourage other teams to measure the noise in other promoters of their own choosing, and allow greater flexibility for future teams in the design and implementation of their genetic networks. We hope that the introduction of this part into the Registry will mark the beginning of a greater appreciation of the value of accounting for stochastic effects on gene expression. </p>
  
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<p>References:</p>
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<p>References UPDATE THESE:</p>
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<p>1: Elowitz, Michael B., et al. "Stochastic gene expression in a single cell." Science 297.5584 (2002): 1183-1186.</p>
 
<p>1: Elowitz, Michael B., et al. "Stochastic gene expression in a single cell." Science 297.5584 (2002): 1183-1186.</p>
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<p>2: Raser, Jonathan M., and Erin K. O'Shea. "Noise in gene expression: origins, consequences, and control." Science 309.5743 (2005): 2010-2013.
 
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Latest revision as of 04:44, 21 November 2015

NOISE - W&M iGEM

Composite Part

Our submission for Best Composite Part is the galK Integrator.

Driven by our insights about the increase in transcriptional noise strength from plasmid copy fluctuations, we decided to provide future teams with a simple way to better manage the noise in the expression of a key protein in their genetic networks by restricting the gene copy fluctuations to within 1 and 2 copies. The galK Integrator is a part which allows easy integration of any sequence onto the galK locus of the E. coli chromosome, which was the locus used by Elowitz et al. to successfully integrate a functional cyan fluorescent protein with an antibiotic resistance cassette [1]. For our method of genome integration 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 below). This product can then be amplified using primers (details found here) and then used in the integration protocol. We have successfully used this part to integrate a 2.1kb segment attached to a 1.1kb antibiotic resistance cassette.

(RIGHT) The galK Integrator will serve to promote the reproducibility of our noise measurements, encourage other teams to measure the noise in other promoters of their own choosing, and allow greater flexibility for future teams in the design and implementation of their genetic networks. We hope that the introduction of this part into the Registry will mark the beginning of a greater appreciation of the value of accounting for stochastic effects on gene expression.


References:

1: Elowitz, Michael B., et al. "Stochastic gene expression in a single cell." Science 297.5584 (2002): 1183-1186.