Team:Uppsala University/Promoters

From 2012.igem.org

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<p style="margin-right:0px;font-size:10px;margin-bottom:10px;float:left;width:400px"><a href="https://static.igem.org/mediawiki/2012/4/49/Promoter_test.png"><img src="https://static.igem.org/mediawiki/2012/4/49/Promoter_test.png" width=400></a><br>
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<p style="margin-right:0px;font-size:10px;margin-bottom:10px;float:left;width:400px"><a href="https://static.igem.org/mediawiki/2012/4/49/Promoter_test.png"><img src="https://static.igem.org/mediawiki/2012/4/49/Promoter_test.png" width=400></a>
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This bar diagram shows the differences measured fluorescence between different promoters in each strand, data from one lab strain should not be compared to the other lab strain since these haven't been grown under exactly same conditions.
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A promoter couldn't be predicted to have the corresponding strength for the screening systems to give a working artificial small RNA / 5´UTR-YFP mRNA ratio, it was necessary to assemble with promoters of various strengths. For this the promoters J23106, J23110 and PlacIQ was chosen.
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To put synthetic and natural promoters on the same scale, a promoter test was carried out. Every promoter was assembled before B0032-SYFP2 (<a href="http://partsregistry.org/Part:BBa_K864101">K864101</a>) in the backbone <a href="http://partsregistry.org/Part:BBa_K592200">K592200</a> (very similar to the pSB3x5 backbones). The test was performed in E coli expression strain MG1655 and cloning strain DH5alpha, by flow cytometry fluorescence measurements of single cells.  
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To prevent future unnecessary assembling by our or any other team a promoter test was conducted on the promoters used in this years iGEM project, a secondary goal of the promoter-test was to map the strengths of the commonly used promoters Plac and PlacIQ.
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Triplicates of each strain and promoter were inoculated in 2 mL LB media with spectinomycin (50 µg/mL) and grown overnight shaking at 37° C. Samples were equilibrated in PBS solution at 1:160 dilution for one hour, and then measured by a BD Biosciences FACSaria III. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Promoter strength is noted as fractions of the reference promoter's, J23101, strength in corresponding strain.  
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The promoter test was conducted by measuring fluorescence expressed by each individual promoter assembled with B0032-SYFP2 (<a href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K864101">BBa_K864101</a>) though a FACS (Fluorescence Activated Cell Sorter) in lab strains MG1655 and DH5α.  
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The T5lac promoter is a hybrid promoter between a promoter from pro-phage T5 and the laci-repressor binding sites from the Plac promoter, so the T5lac promoter has the strength of the T5 pro-phage promoter but can be repressed by the laci-repressor.
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The test was conducted on triplicated cultures of each promoter construct, which were measured in the FACS. For each culture 10⁵ cells fluorescence were measured individually, dead cells and non fluorescent cells were discarded, and an average was calculated. All promoter constructs of each strain were grown under same conditions.
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The T5lac promoter is a hybrid promoter between a promoter from pro-phage T5 and the laci-repressor binding sites from the Plac promoter, so the T5lac promoter has the strength of the T5 pro-phage promoter but can be repressed by the laci-repressor.
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<td><b>MG1566 </b></td>
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<td><b>DH5alpha </b></td>
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<td>J23101 </td>
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<td>1 </td>
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<td>1 </td>
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<td>J23106 </td>
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<td>0.19 </td>
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<td>0.37</td>
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<td>J23110 </td>
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<td>0.27 </td>
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<td>0.50 </td>
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<td>J23116 </td>
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<td>N/A</td>
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<td>0.11</td>
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<td>Plac</td>
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<td>0.34</td>
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<td>0.67</td>
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<td>PlacIq</td>
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<td>0.03</td>
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<td>0.05</td>
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<td>T5lac</td>
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<td>0.217</td>
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<td>0.54</td>
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<td>PLlacO</td>
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<td>0.87</td>
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<td>N/A</td>
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The variance in expression between MG1655 and DH5α may depend on the J23101-B0032-SYFP2, which is the promoter used as reference. The construct with J23101 in DH5α may have been a weaker than average clone, resulting in higher RPU values compared to other DH5α.
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The variance in expression between MG1655 and DH5α may depend on the reference J23101. The maximum protein expression may be lower in DH5α, due to its lower fitness resulting in lower expression of SYFP2 in the J23101 construct. Alternatively, the clone with J23101 in DH5α may have been weaker than average, resulting in higher RPU values compared to other DH5α.  
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Alternatively the maximum protein expression is lower in DH5α due to its lower fitness also resulting in lower expression of SYFP2 in the J23101 construct.
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Revision as of 17:01, 18 October 2012

Team Uppsala University – iGEM 2012


Promoter Tests

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To put synthetic and natural promoters on the same scale, a promoter test was carried out. Every promoter was assembled before B0032-SYFP2 (K864101) in the backbone K592200 (very similar to the pSB3x5 backbones). The test was performed in E coli expression strain MG1655 and cloning strain DH5alpha, by flow cytometry fluorescence measurements of single cells.

Triplicates of each strain and promoter were inoculated in 2 mL LB media with spectinomycin (50 µg/mL) and grown overnight shaking at 37° C. Samples were equilibrated in PBS solution at 1:160 dilution for one hour, and then measured by a BD Biosciences FACSaria III. 10^5 cells of each sample were individually measured and averaged, with dead and other non-flourescent cells excluded. Promoter strength is noted as fractions of the reference promoter's, J23101, strength in corresponding strain.

The T5lac promoter is a hybrid promoter between a promoter from pro-phage T5 and the laci-repressor binding sites from the Plac promoter, so the T5lac promoter has the strength of the T5 pro-phage promoter but can be repressed by the laci-repressor.

MG1566 DH5alpha
J23101 1 1
J23106 0.19 0.37
J23110 0.27 0.50
J23116 N/A 0.11
Plac 0.34 0.67
PlacIq 0.03 0.05
T5lac 0.217 0.54
PLlacO 0.87 N/A

The variance in expression between MG1655 and DH5α may depend on the reference J23101. The maximum protein expression may be lower in DH5α, due to its lower fitness resulting in lower expression of SYFP2 in the J23101 construct. Alternatively, the clone with J23101 in DH5α may have been weaker than average, resulting in higher RPU values compared to other DH5α.



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