Team:Glasgow/Results/PromoterLibrary

From 2011.igem.org

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{{Team:Glasgow/Header}}
{{Team:Glasgow/Header}}
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<html><h1>Promoter Library & Multiple Cloning Site</h1>
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<html><h1>Promoter Library</h1>
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<h6><a href="https://2011.igem.org/Team:Glasgow/Results">Back to Results</a></h6>
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<h3>Aims:</h3>
<h3>Aims:</h3>
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The aims of creating a new promoter/RBS library are:<br/><br/>
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The aims of creating a promoter/RBS library are:<br/><br/>
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1. To transform and miniprep a stock of promoters<br/>
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- To create a new tool set for controlling gene expression levels in a modular pathway<br/>
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2. To transform and miniprep a strong and weak ribosome binding site<br/>
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- To increase the efficiency of construct production by reducing the total number of ligations to be performed
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3. To ligate a variety of promoters to a strong ribosome binding site<br/>
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4. To ligate a variety of promoters to a weak ribosome binding site<br/>
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5. To show quicker and fuller characterisation of promoter and ribosome binding site combinations<br/>
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<center><img src=https://static.igem.org/mediawiki/2011/d/d4/Glasgowprom%2BRbs.png></center>
<h3>Methods:</h3>
<h3>Methods:</h3>
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<b><u>A Stock of Promoters and RBS</b></u><br/>
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<b><u>A Stock of Promoters and RBS</b></u><br/><br/>
1. Transform <i>Top 10</i> with DNA from the kit plate.<br/><br/> The following Promoters were chosen:<br/>
1. Transform <i>Top 10</i> with DNA from the kit plate.<br/><br/> The following Promoters were chosen:<br/>
Strong Promoter <a href=http://partsregistry.org/Part:BBa_J23119>(BBa_J23119)</a><br/>
Strong Promoter <a href=http://partsregistry.org/Part:BBa_J23119>(BBa_J23119)</a><br/>
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OmpF Promoter <a href=http://partsregistry.org/Part:BBa_R0084> (BBa_R0084)</a><br/><br/>
OmpF Promoter <a href=http://partsregistry.org/Part:BBa_R0084> (BBa_R0084)</a><br/><br/>
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The following Ribosome Binding Sites were chosen:<br/>
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The following Ribosome Binding Sites were chosen:<br/><br/>
Strong RBS <a href=<a href=http://partsregistry.org/Part:BBa_B0034>(BBa_B0034)</a><br/>
Strong RBS <a href=<a href=http://partsregistry.org/Part:BBa_B0034>(BBa_B0034)</a><br/>
Weak RBS <a href=<a href=http://partsregistry.org/Part:BBa_J61101>(BBa_J61101)</a><br/><br/>
Weak RBS <a href=<a href=http://partsregistry.org/Part:BBa_J61101>(BBa_J61101)</a><br/><br/>
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6. Transform the ligation into <i>E.coli.</i> Overnight and miniprep colonies.<br/>
6. Transform the ligation into <i>E.coli.</i> Overnight and miniprep colonies.<br/>
<br/><center><img src=https://static.igem.org/mediawiki/2011/7/7c/AlwnImethod.png><br/></center>
<br/><center><img src=https://static.igem.org/mediawiki/2011/7/7c/AlwnImethod.png><br/></center>
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<h3>Results:</h3>
 
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<b><u>Stock of Promoters and Ribosome Binding Sites</b></u>
 
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We have successfully created a stock of all the promoters and ribosome binding sites which we aimed to make.
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Click <a href=https://2011.igem.org/Team:Glasgow/Results/PromoterLibrary/Results>here</a> for results!<br/>
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<br/><br/>
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<b><u>Stocks of Ligated Constructs</b></u>
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We have successfully obtained colonies from the following ligations:
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Strong Promoter + Strong RBS<Br/>
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Strong Promoter + Weak RBS<br/>
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Weak Promoter + Strong RBS<br/>
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Weak Promoter + Weak RBS<br/>
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pBAD Promoter + Strong RBS<br/>
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pBAD promoter + Weak RBS<br/>
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Bluf Promoter + Strong RBS<br/>
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Bluf Promoter + Weak RBS<br/>
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OmpF Promoter + Strong RBS<br/>
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OmpF Promoter + Weak RBS<br/>
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OmpC Promoter + Strong RBS<br/>
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OmpC Promoter + Weak RBS <br/><br/>
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<img src=https://static.igem.org/mediawiki/2011/1/1d/Glasgowgel_-_bluf_%2B_prom.png align=left><br/>
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<br/>These parts are all present in the plasmid which the promoter part was present in on the Registry.<br/>Upon ligating the parts into the submission vector (pSB1C3), only the following constructs successfully grew and were confirmed to be the right size on a gel:<br/>
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Bluf Promoter + Strong RBS<br/>
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Bluf Promoter + Weak RBS<br/>
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OmpF + Weak RBS <br/>
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pBAD + Strong RBS<br/>
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Weak Promoter + Strong RBS<br/>
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Strong Promoter + Strong RBS<br/>
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Weak Promoter + Weak RBS<br/><br/><br/><br/><br/><br/><br/><br/>
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<b><u>Gel of Weak Promoter + Strong RBS</b></u><br/><Br/>
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<img src=https://static.igem.org/mediawiki/2011/3/39/Glasgowgel_-_weak_prom_%2B_s.rbs.png><br/><br/>
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<b><u>Gel of Weak Promoter + Weak RBS</b></u><br/><br/>
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<img src=https://static.igem.org/mediawiki/2011/d/db/Glasgowgel_-_w.prom_%2B_w.rbs.png><br/><br/>
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<b><u>Gel of OmpF + Strong RBS and OmpF + Weak RBS</b></u><br/><br/>
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<img src=https://static.igem.org/mediawiki/2011/4/4f/Glasgowgel_-_ompf.png><br/><br/>
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<h2>Multiple Cloning Site</h2>
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<b><u>Aim:</b></u><br/>
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- To create a less time consuming and more efficient method of testing gene constructs
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<b><u>Method</b></u>
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We have designed a novel biobrick with a Multiple Cloning Site. <br/>The restriction enzymes we chose to include are XhoI, BamHI, SalI, BglI and HindIII. These are not compatible with biobrick restriction sites and so will allow for testing of gene constructs before the prefix and suffix have been added.<br/>
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<img src=https://static.igem.org/mediawiki/2011/1/10/GlasgowMCSbiobrick.jpg><br/><br/>This can be used for more efficient testing of biobricks and will reduce the number of overall ligations a team must carry out.  <br/>The <br/>
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<b><u>Results</b></u><br/><br/>
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The part is currently awaiting synthesis. <br/>
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Latest revision as of 03:52, 22 September 2011

Promoter Library

Back to Results

Aims:

The aims of creating a promoter/RBS library are:

- To create a new tool set for controlling gene expression levels in a modular pathway
- To increase the efficiency of construct production by reducing the total number of ligations to be performed

Methods:

A Stock of Promoters and RBS

1. Transform Top 10 with DNA from the kit plate.

The following Promoters were chosen:
Strong Promoter (BBa_J23119)
Weak Promoter (BBa_J23106)
pBAD Promoter (BBa_K206000)
Bluf Promoter (BBa_K238013)
OmpC Promoter (BBa_R0082)
OmpF Promoter (BBa_R0084)

The following Ribosome Binding Sites were chosen:

Strong RBS (BBa_B0034)
Weak RBS (BBa_J61101)

2. Overnight the successful colonies
3. Miniprep the overnights

A Stock of Constructs of Promoters with RBS

We aimed to make the following constructs:

Strong Promoter + Strong RBS
Strong Promoter + Weak RBS
Weak Promoter + Strong RBS
Weak Promoter + Weak RBS
pBAD Promoter + Strong RBS
pBAD Promoter + Weak RBS
Bluf Promoter + Strong RBS
Bluf Promoter + Weak RBS
OmpC Promoter + Strong RBS
OmpC Promoter + Weak RBS

The AlwnI Method
Both Ribosome Binding Sites that we worked with are only 12 base pairs long, making them impossible to visalise on an agarose gel.
This is a common problem when ligating small Biobrick parts together. For this reason we had to come up with a novel method of ligating these small constructs. This method, the ''AlwnI'' method, uses the restriction enzyme "AlwnI" to cut plasmids in a site which is commonly found in origins of replication. It is detailed below.

1. Digest the plasmid containing the promoter with AlwnI and SpeI.
2. Digest the plasmid containing the ribosome binding site with AlwnI and XbaI.
3. Run the samples on a gel.
4. Perform a gel extraction. Ensure you know the size of the fragments you are keeping.
5. Ligate the promoter fragment to the RBS fragment.
6. Transform the ligation into E.coli. Overnight and miniprep colonies.



Click here for results!

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