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!

@@ Line 1: / Line 1: @@
 {{Team:Glasgow/Header}}
-<html><h1>Promoter Library & Multiple Cloning Site</h1>
+<html><h1>Promoter Library</h1>
+<h6><a href="https://2011.igem.org/Team:Glasgow/Results">Back to Results</a></h6>
 <h3>Aims:</h3>
-The aims of creating a new promoter/RBS library are:<br/><br/>
+The aims of creating a promoter/RBS library are:<br/><br/>
-. To transform and miniprep a stock of promoters<br/>
+- To create a new tool set for controlling gene expression levels in a modular pathway<br/>
-. To transform and miniprep a strong and weak ribosome binding site<br/>
+- To increase the efficiency of construct production by reducing the total number of ligations to be performed
-. To ligate a variety of promoters to a strong ribosome binding site<br/>
-. To ligate a variety of promoters to a weak ribosome binding site<br/>
-. To show quicker and fuller characterisation of promoter and ribosome binding site combinations<br/>
+<center><img src=https://static.igem.org/mediawiki/2011/d/d4/Glasgowprom%2BRbs.png></center>
 <h3>Methods:</h3>
-<b><u>A Stock of Promoters and RBS</b></u><br/>
+<b><u>A Stock of Promoters and RBS</b></u><br/><br/>
 . 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/>
@@ Line 22: / Line 22: @@
 OmpF Promoter <a href=http://partsregistry.org/Part:BBa_R0084> (BBa_R0084)</a><br/><br/>
-The following Ribosome Binding Sites were chosen:<br/>
+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/>
 Weak RBS <a href=<a href=http://partsregistry.org/Part:BBa_J61101>(BBa_J61101)</a><br/><br/>
@@ Line 56: / Line 56: @@
 . 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/>
+Click <a href=https://2011.igem.org/Team:Glasgow/Results/PromoterLibrary/Results>here</a> for results!<br/>
-<h3>Results:</h3>
-We have successfully obtained colonies from the following ligations:
-<br/><br/>
-Strong Promoter + Strong RBS<Br/>
-Strong Promoter + Weak RBS<br/>
-Weak Promoter + Strong RBS<br/>
-Weak Promoter + Weak RBS<br/>
-pBAD Promoter + Strong RBS<br/>
-pBAD promoter + Weak RBS<br/>
-Bluf Promoter + Strong RBS<br/>
-Bluf Promoter + Weak RBS<br/>
-OmpF Promoter + Strong RBS<br/>
-OmpF Promoter + Weak RBS<br/>
-OmpC Promoter + Strong RBS<br/>
-OmpC Promoter + Weak RBS <br/><Br/><br/>
-However, all the parts were in the original plasmid from the kit. We digested and ligated them to the submission vector, pSB1C3. Only the following constructs successfully grew and were confirmed to be the right size on a gel<br/>
-Bluf Promoter + Strong RBS<br/>
-Bluf Promoter + Weak RBS<br/>
-OmpF + Weak RBS <br/>
-pBAD + Strong RBS<br/>
-Weak Promoter + Strong RBS<br/>
-Strong Promoter + Strong RBS<br/>
-Weak Promoter + Weak RBS<br/>
-Bluf Promoter + RBS + RFP<br/><br/><Br/>
-The next step was to ligate RFP to the constructs in order to test them.  However at this step, only the following colonies successfully grew:</br>
-OmpC + Strong RBS + RFP <Br/>
-OmpC + Weak RBS + RFP<br/>
-OmpF + Strong RBS + RFP<br/>
-OmpF + Weak RBS +RFP <br/>
-<br/><br/>
-We suspect a number of potential reasons for this lack of growth.  There were initially problems trying to transform the RFP <a href=http://partsregistry.org/Part:BBa_B0034>(Part:E1010).</a> We also cannot check if the RBS has definitely ligated to the promoter, although we believe it has due to using the AlwnI method described above and gel extracting to take the right fragments.<br/><br/>