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From 2011.igem.org

Promoter Library & Multiple Cloning Site

Aims

The aims of creating a new promoter/RBS library are:

To transform and miniprep a stock of promoters
To transform and miniprep a strong and weak ribosome binding site
To ligate a variety of promoters to a strong ribosome binding site
To ligate a variety of promoters to a weak ribosome binding site
To develop a new way of assembling promoters and ribosome binding sites.
To allow for quicker and fuller characterisation of promoters
To shorten the amount of time that is spent ligating of constructs

Methods

We aimed to make the following constructs:

Strong Promoter (BBa_J23119) + Strong RBS(BBa_B0034)
Strong Promoter (BBa_J23119) + Weak RBS (BBa_J61101)
Weak Promoter (BBa_J23106)+ Strong RBS (BBa_B0034)
Weak Promoter (BBa_J23106) + Weak RBS (BBa_J61101)
pBAD Promoter (BBa_K206000) + Strong RBS (BBa_B0034)
pBAD Promoter (BBa_K206000) + Weak RBS (BBa_J61101)
Bluf Promoter (BBa_K238013) + Strong RBS(BBa_B0034)
Bluf Promoter (BBa_K238013) + Weak RBS (BBa_J61101)
OmpC Promoter (BBa_R0082) + Strong RBS(BBa_B0034)
OmpC Promoter (BBa_R0082) + Weak RBS (BBa_B0034)
OmpF Promoter (BBa_R0084) + Strong RBS(BBa_B0034)
OmpF Promoter (BBa_R0084) + Weak RBS (BBa_B0034)
We selected two Ribosome Binding sites of different strength from the Registry. These were a Strong Ribosome Binding Site (Part:BBa_B0034) and a Weak Ribosome Binding Site (Part:BBa_J61101). Both these parts are only 12 base pairs long, making them impossible to visalise on a 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. 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.

Results

Bluf Promoter + Strong RBS
Bluf Promoter + Weak RBS
OmpF + Weak RBS
pBAD + Strong RBS
Weak Promoter + Strong RBS
Strong Promoter + Strong RBS
Weak Promoter + Weak RBS
Bluf Promoter + RBS + RFP
Develop a new way of assembling library of promoters and ribosome binding sites easier biobricks better characterisation of promoters more time to work on genes/characterise by simplying the ligation process