Difference between revisions of "Part:BBa K1741005"

Revision as of 06:53, 27 September 2015

sfGFP under rhamnose promoter with removed EcoRI site

RhamnoseWT promoter has been copied from E. coli genome: ggtgagcatcacatcaccacaattcagcaaattgtgaacatcatcacgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaaggtcgcGAATTCaggcgctttttagactggtcgtaatgaaattcagcaggatcacatt then Eco RI site was removed by PCR to obtain Rha1 in a biobrick standard: ggtgagcatcacatcaccacaattcagcaaattgtgaacatcatcacgttcatctttccctggttgccaatggcccattttcctgtcagtaacgagaaggtcgcGAATTTaggcgctttttagactggtcgtaatgaaattcagcaggatcacatt The only one point mutation makes the promoter slightly stronger and more sensitive to rhamnose.

Design

Legend:

RhaWT - rhamnose wild type

Rha1 - [BBa_K1741005]

Rha2 - [BBa_K1741006]

Our wild type rhamnose promoter is commercially available. However, we had to modify it to remove EcoRI restriction site to fit BioBrick standards. We achieved it by a single point mutation of cytosine to thymine - that is how Rha1 was created. Next, we decided to further minimize the promoter sequence by removing the CRP site - we called this contstruct Rha2.

Characteristic and Results

Legend:

RhaWT - rhamnose wild type

Rha1 - [BBa_K1741005]

Rha2 - [BBa_K1741006]

The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.

All three rhamnose promoters are induced by rhamnose and repressed by glucose. Turns out that single point mutation in EcoRI restriction site in Rha1 significantly increased the activity of the promoter as comapred to the wild type construct. Rha2 is a weak promoter, perfect for people who would like to manufacture small amount of the protein of interest.

We also checked the tightness of our promoters.

All of our promoters are induced only by their respective sugars with a small exception of xylose promoters being slightly induced by arabinose and arabinose promoters being slightly induced by xylose. All of our promoters are repressed by glucose.

Sequence and Features