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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<h3>Aims</h3>
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<h6><a href="https://2011.igem.org/Team:Glasgow/Results">Back to Results</a></h6>
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The aims of creating a new promoter/RBS library are:<br/>
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To ligate a variety of promoters to a strong ribosome binding site<br/>
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To ligate a variety of promoters to a weak ribosome binding site<br/>
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To develop a new way of assembling promoters and ribosome binding sites.  <br/>
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To allow for quicker and fuller characterisation of promoters<br/>
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To shorten the amount of time that is spent on ligation of constructs<br/>
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<h3>Aims:</h3>
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The aims of creating a promoter/RBS library are:<br/><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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- To increase the efficiency of construct production by reducing the total number of ligations to be performed
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<h3>Methods</h3>
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<center><img src=https://static.igem.org/mediawiki/2011/d/d4/Glasgowprom%2BRbs.png></center>
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We selected two Ribosome Binding sites of different strength from the Registry. These were a Strong Ribosome Binding Site <a href=http://partsregistry.org/Part:BBa_B0034>(Part:BBa_B0034)</a> and a Weak Ribosome Binding Site <a href=http://partsregistry.org/Part:BBa_J61101>(Part:BBa_J61101).</a> Both these parts are only 12 base pairs long, making them impossible to visalise on a gel.<br/>
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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.  It is detailed below.<br/><br/>
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<h3>Methods:</h3>
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1. Digest the plasmid containing the promoter with AlwnI and SpeI.<br/>
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2. Digest the plasmid containing the ribosome binding site with AlwnI and XbaI.</br>
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3. Run the samples on a gel.<br/>
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<b><u>A Stock of Promoters and RBS</b></u><br/><br/>
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4. Perform a gel extraction. Ensure you know the size of the fragments you are keeping.<br/>
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1. Transform <i>Top 10</i> with DNA from the kit plate.<br/><br/> The following Promoters were chosen:<br/>
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5. Ligate the promoter fragment to the RBS fragment.<br/><br/>
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Strong Promoter <a href=http://partsregistry.org/Part:BBa_J23119>(BBa_J23119)</a><br/>
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<img src=https://static.igem.org/mediawiki/2011/7/7c/AlwnImethod.png><br/>
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Weak Promoter <a href=http://partsregistry.org/Part:BBa_J23106>(BBa_J23106)</a><br/>
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pBAD Promoter <a href=http://partsregistry.org/Part:BBa_K206000> (BBa_K206000)</a><br/>
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Bluf Promoter <a href=http://partsregistry.org/Part:BBa_K238013> (BBa_K238013)</a><br/>
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OmpC Promoter <a href=http://partsregistry.org/Part:BBa_R0082> (BBa_R0082)</a><br/>
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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/><br/>
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Strong RBS <a href=<a href=http://partsregistry.org/Part:BBa_B0034>(BBa_B0034)</a><br/>
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Weak RBS <a href=<a href=http://partsregistry.org/Part:BBa_J61101>(BBa_J61101)</a><br/><br/>
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2. Overnight the successful colonies<br/>
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3. Miniprep the overnights<br/><br/>
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<b><u>A Stock of Constructs of Promoters with RBS</b></u>
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<br/><br/>
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We aimed to make the following constructs:</br><br/>
Strong Promoter + Strong RBS<br/>
Strong Promoter + Strong RBS<br/>
Strong Promoter + Weak RBS<br/>
Strong Promoter + Weak RBS<br/>
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Bluf Promoter + Strong RBS<br/>
Bluf Promoter + Strong RBS<br/>
Bluf Promoter + Weak RBS<br/>
Bluf Promoter + Weak RBS<br/>
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OmpC Promoter + Strong RBS<Br/>
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OmpC Promoter + Strong RBS<br/>
OmpC Promoter + Weak RBS<br/>
OmpC Promoter + Weak RBS<br/>
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OmpF Promoter + Strong RBS<Br/>
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<br/>
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OmpF Promoter + Weak RBS<br/>
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<h3>Results</h3>
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<b><u>The AlwnI Method</b></u><br/>
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Bluf Promoter + Strong RBS<br/>
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Both Ribosome Binding Sites that we worked with are only 12 base pairs long, making them impossible to visalise on an agarose gel.<br/>
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Bluf Promoter + Weak RBS<br/>
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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.<br/><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/>
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Bluf Promoter + RBS + RFP<br/>
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1. Digest the plasmid containing the promoter with AlwnI and SpeI. <br/>
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2. Digest the plasmid containing the ribosome binding site with AlwnI and XbaI.</br>
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Develop a new way of assembling library of promoters and ribosome binding sites
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3. Run the samples on a gel.<br/>
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easier biobricks
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4. Perform a gel extraction. Ensure you know the size of the fragments you are keeping.<br/>
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better characterisation of promoters
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5. Ligate the promoter fragment to the RBS fragment.<br/>
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more time to work on genes/characterise by simplying the ligation process
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6. Transform the ligation into <i>E.coli.</i> Overnight and miniprep colonies.<br/>
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<br/><center><img src=https://static.igem.org/mediawiki/2011/7/7c/AlwnImethod.png><br/></center>
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<br/>
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Click <a href=https://2011.igem.org/Team:Glasgow/Results/PromoterLibrary/Results>here</a> for results!<br/>

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!

Retrieved from "http://2011.igem.org/Team:Glasgow/Results/PromoterLibrary"