Difference between revisions of "Team:Austin UTexas LASA/Contribution"
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| − | <h3> | + | <h3>Group: Austin_UTexas_LASA 2017</h3> |
| − | <p>< | + | <p>Summary: We validated the promoter function by a fluorescence detection assay utilizing green fluorescent protein (GFP)-fused construct to BBa_I14034 promoter part.</p> |
| − | < | + | <br></br> |
| − | < | + | <p><b> |
| − | + | This construct is produced by combining BBa_114034 promoter part and a GFP coding sequence. This plasmid contains ColE origin and CamR resistance. | |
| − | + | <br></br> | |
| + | Protocol | ||
| + | <ol> | ||
| + | <li>Liquid cultures</li> | ||
| + | <li>Pick colonies from plate</li> | ||
| + | <li>Multiple controls </li> | ||
| + | <li>Overnight in the 37 incubator </li> | ||
| + | <li>Liquid cultures of already grown liquid cultures</li> | ||
| + | <li>100 uL of grown liquid cultures into 5 mL of media + CamR</li> | ||
| + | <li>Incubate cultures for another 4-6 hours </li> | ||
| + | <li>Spin down cultures </li> | ||
| + | <li>3000 rpm for 5 minutes</li> | ||
| + | <li>Resuspend in 1x Buffer</li> | ||
| + | <li>4 mL of 1x PBS </li> | ||
| + | <li>Pipette or vortex to resuspend cells</li> | ||
| + | <li>Plate reading</li> | ||
| + | <li>Take 100 uL of the resuspended cells</li> | ||
| + | <li>Load into a 96 well transparent plate</li> | ||
| + | <li>Measure absorbance and fluorescence with GFP assay (excitation: 495 nm, emission: 509 nm) | ||
| + | </li> | ||
| + | </ol> | ||
| + | Three negative control constructs containing different promoter sequences were used and compared in this assay: 1) T7 promoter (Invitrogen, USA), 2) pCP25 (source, ) and 3) pPA3 promoter (source, ). These three promoter do not contain GFP coding sequence. | ||
| + | <br></br> | ||
| + | For the fluorescence detection, we used a plate reader ( ) which is equipped in the Ellington lab in UT Austin. | ||
| + | <br></br> | ||
| + | Because the part’s description stated that part BBa_I14034 is designed as a constitutive promoter, we did not add Isopropyl b-D-1-thiogalactopyranoside (IPTG) as a inducer for gene expression. | ||
| + | <br></br> | ||
| + | The plate reader measured the absorbance and fluorescence of each cell containing the BBa_I14034::GFP construct and non-GFP construct. Value of fluorescence was divided by cell density (absorbance) and the final result was shown as a graph in Figure 1 (below). The graph in figure 2 (below) shows a close-view of the fluorescence measured of the GFP driven by the control promoters. | ||
| + | <br></br> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/b/ba/T--Austin_UTexas_LASA--promotercontrols.png"> | ||
| + | <img src="https://static.igem.org/mediawiki/2017/1/10/T--Austin_UTexas_LASA--driv.png"> | ||
| + | <br></br> | ||
| + | RFU (relative fluorescence units) = value of fluorescence/value of absorbance | ||
| + | <br></br> | ||
| + | The result demonstrates that BBa_I14034 promoter is working to expression GFP in bacterial cells. Level of GFP expression was around 9,500 RFU. Cells containing non-GFP controls (T7::empty construct, CP25::empty construct, and PA3 empty construct) did not show significant detections. | ||
| + | <br></br> | ||
| + | <br></br> | ||
| + | <img src ="https://static.igem.org/mediawiki/2017/6/6f/T--Austin_UTexas_LASA--contrib.jpg" width="50%" height = "50%" align = "right" style="margin:5px 5px"> | ||
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Revision as of 03:09, 2 November 2017
Contribution
Group: Austin_UTexas_LASA 2017
Summary: We validated the promoter function by a fluorescence detection assay utilizing green fluorescent protein (GFP)-fused construct to BBa_I14034 promoter part.
This construct is produced by combining BBa_114034 promoter part and a GFP coding sequence. This plasmid contains ColE origin and CamR resistance.
Protocol
Three negative control constructs containing different promoter sequences were used and compared in this assay: 1) T7 promoter (Invitrogen, USA), 2) pCP25 (source, ) and 3) pPA3 promoter (source, ). These three promoter do not contain GFP coding sequence.
For the fluorescence detection, we used a plate reader ( ) which is equipped in the Ellington lab in UT Austin.
Because the part’s description stated that part BBa_I14034 is designed as a constitutive promoter, we did not add Isopropyl b-D-1-thiogalactopyranoside (IPTG) as a inducer for gene expression.
The plate reader measured the absorbance and fluorescence of each cell containing the BBa_I14034::GFP construct and non-GFP construct. Value of fluorescence was divided by cell density (absorbance) and the final result was shown as a graph in Figure 1 (below). The graph in figure 2 (below) shows a close-view of the fluorescence measured of the GFP driven by the control promoters.
RFU (relative fluorescence units) = value of fluorescence/value of absorbance
The result demonstrates that BBa_I14034 promoter is working to expression GFP in bacterial cells. Level of GFP expression was around 9,500 RFU. Cells containing non-GFP controls (T7::empty construct, CP25::empty construct, and PA3 empty construct) did not show significant detections.
