Difference between revisions of "Team:William and Mary/Description"
Line 159: | Line 159: | ||
Original work done by Michael Elowitz investigated stochasticity in gene expression at the single cell level using a dual-reporter fluorescent system [1]. In this method two constructs are created that both drive expression of a fluorescent protein using the same promoter, RBS, and terminator. One of these reporter constructs uses YFP and the other CFP. This dual reporter system is what allows the investigator to differentiate between the extrinsic factors of noise, which should affect both constructs equally, and the intrinsic factors, which will result in a difference in the ratio of CFP: YFP fluorescence. These constructs are then integrated (notebook link here) into specific sites on the genome and the fluorescence output measured using fluorescent imaging. </p> <p> | Original work done by Michael Elowitz investigated stochasticity in gene expression at the single cell level using a dual-reporter fluorescent system [1]. In this method two constructs are created that both drive expression of a fluorescent protein using the same promoter, RBS, and terminator. One of these reporter constructs uses YFP and the other CFP. This dual reporter system is what allows the investigator to differentiate between the extrinsic factors of noise, which should affect both constructs equally, and the intrinsic factors, which will result in a difference in the ratio of CFP: YFP fluorescence. These constructs are then integrated (notebook link here) into specific sites on the genome and the fluorescence output measured using fluorescent imaging. </p> <p> | ||
− | <div><p><IMG SRC="https://static.igem.org/mediawiki/2015/e/ed/WmRaserHoriz.jpeg"></p><p> Panel A shows an example of intrinsic and extrinsic noise; both the ratio of the fluorescence of the two reporters (intrinsic) and the absolute fluorescence of the two reporters (extrinsic) changes. Panel B shows an example of extrinsic noise only; the ratio of the fluorescence of the two reporters stays constant while the absolute fluorescence changes | + | <div><p><IMG SRC="https://static.igem.org/mediawiki/2015/e/ed/WmRaserHoriz.jpeg"></p><p>(ABOVE) Panel A shows an example of intrinsic and extrinsic noise; both the ratio of the fluorescence of the two reporters (intrinsic) and the absolute fluorescence of the two reporters (extrinsic) changes. Panel B shows an example of extrinsic noise only; the ratio of the fluorescence of the two reporters stays constant while the absolute fluorescence changes [2].</p></div></p> |
<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> | <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> | ||
<div><p style="float: right;"><IMG SRC="https://static.igem.org/mediawiki/2015/9/97/WMdCasDiag.jpeg" width=600px></p><p> | <div><p style="float: right;"><IMG SRC="https://static.igem.org/mediawiki/2015/9/97/WMdCasDiag.jpeg" width=600px></p><p> | ||
− | Schematic of dCas9/CRISPR system. gRNAs targeting the promoter region of the gene of interest will guide dCas9 binding. Bound dCas9 sterically hinders RNAP and transcription factors from binding and promoting transcription.</p> | + | (RIGHT)Schematic of dCas9/CRISPR system. gRNAs targeting the promoter region of the gene of interest will guide dCas9 binding. Bound dCas9 sterically hinders RNAP and transcription factors from binding and promoting transcription [1].</p> |
<p>In addition to characterizing the noise of promoters in iGEM, we contributed additional tools for manipulating their expression. CRISPR/Cas9 has been used extensively in synthetic biology, both inside and out of iGEM. In particular, new functionalizations of the Cas9 protein that remove its catalytic nuclease domain while retaining its DNA-binding activity have allowed for novel methods in molecular biology. This catalytically inactive variant of Cas9, known as dCas9, can be used to repress gene expression by targeting the promoter region of a gene of interest. This repression is mediated by the CRISPR/Cas9 complex binding to the promoter region and can block RNAP binding or prevent transcriptional elongation. All of our gRNAs prevent RNAP binding and initiation of transcription.</p></div></p> | <p>In addition to characterizing the noise of promoters in iGEM, we contributed additional tools for manipulating their expression. CRISPR/Cas9 has been used extensively in synthetic biology, both inside and out of iGEM. In particular, new functionalizations of the Cas9 protein that remove its catalytic nuclease domain while retaining its DNA-binding activity have allowed for novel methods in molecular biology. This catalytically inactive variant of Cas9, known as dCas9, can be used to repress gene expression by targeting the promoter region of a gene of interest. This repression is mediated by the CRISPR/Cas9 complex binding to the promoter region and can block RNAP binding or prevent transcriptional elongation. All of our gRNAs prevent RNAP binding and initiation of transcription.</p></div></p> | ||
Revision as of 11:51, 18 September 2015
Integrator Suites
Detail1
Detail2
Detail3
Detail4
text
text
Antibiotic Operons
Detail1
Detail2
Detail3
Detail4
text
text
dCas9s
Detail1
Detail2
Detail3
Detail4
text
text
gRNAs
Detail1
Detail2
Detail3
Detail4
text
text
XFPs Under Various Promoters
Detail1
Detail2
Detail3
Detail4
text
text
G^2
Detail1
Detail2
Detail3
Detail4
text
text