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From 2011.igem.org

Promoter Library & Multiple Cloning Site

Aims:

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

1. To transform and miniprep a stock of promoters
2. To transform and miniprep a strong and weak ribosome binding site
3. To ligate a variety of promoters to a strong ribosome binding site
4. To ligate a variety of promoters to a weak ribosome binding site
5. To develop a new way of assembling promoters and ribosome binding sites.
6. To allow for quicker and fuller characterisation of promoters
7. To shorten the amount of time that is spent ligating of constructs
8. To develop a new, more efficient method for testing gene constructs

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 (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.
6. Transform the ligation into E.coli. Overnight and miniprep colonies.
7. Digest and ligate RFP to the promoter and RBS construct.

Results:

We have successfully obtained colonies from the following ligations:

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
OmpF Promoter + Strong RBS
OmpF Promoter + Weak RBS
OmpC Promoter + Strong RBS
OmpC Promoter + Weak RBS


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
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


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:
OmpC + Strong RBS + RFP
OmpC + Weak RBS + RFP
OmpF + Strong RBS + RFP
OmpF + Weak RBS +RFP


We suspect a number of potential reasons for this lack of growth. There were initially problems trying to transform the RFP (Part:E1010). 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.