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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 show quicker and fuller characterisation of promoter and ribosome binding site combinations

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.

Results:

Stock of Promoters and Ribosome Binding Sites
We have successfully created a stock of all the promoters and ribosome binding sites which we aimed to make.

Stocks of Ligated Constructs

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



These parts are all present in the plasmid which the promoter part was present in on the Registry.
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:
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

Gel of Weak Promoter + Strong RBS



Gel of Weak Promoter + Weak RBS



Gel of OmpF + Strong RBS and OmpF + Weak RBS