Reporter

Part:BBa_K1311007:Experience

Designed by: Luning Yang   Group: iGEM14_Tsinghua-A   (2014-10-09)

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Applications of BBa_K1311007

This parts can convert the binding ability of TALE protein to its target DNA sequence to a easier available parameter, the florescent intensity of RFP. When the TALE protein is expressed, the TALE make binds to its target, which may interrupt the transcription of LacI. The lack of repressors may lead to the expression of RFP. So the stronger florescent intensity means the better binding ability of TALE protein.

Validation
We change the normal RBS sequence in the LacI coding sequence into an RBS containing three tandem TALE binding site sequences. So we have to validate that the LacI protein can express normally and can normally inhibit the expression of RFP.
We transformed this plasmid in to E.coli DH5α with electroporation and chemical transformation. We spread the plate and culture it in 37 degrees Centigrade for more than 15 hours until small colonies can be seen in the plate. At this moment, the colonies might look red for the sake of the delay of expression of LacI. We picked colonies into the 10mL tubes and added 5mL LB broth with Chl antibiotics. After six hours’ shaking at 37 degrees Centigrade and 220 rpm, we got the tubes out and double, four times, eight times diluted. (40μL of 0.1M IPTG was added as the positive control group) After another shaking for 12h, florescence of the bacteria was evaluated with an enzyme-labeled instrument and OD600 was tested with spectrometry.

Results are shown below.


TsinghuaaParts2.jpg


Picture of our tubes
(The two red ones on the back are the positive control groups, others are not red)
Figures about the OD600 and Flurescence Intensity of the broth after 12h shaking


Reportfigure_TsinghuaA.jpg


The first column with IPTG as positive control. The FI of the positive control is much higher than the others, which means our LacI works normally.

Test binding ability of TALE protein with report system.
Integrate the constitutive TALE protein express cassette(BBa_K1311005) with 18-repeat TALE protein(BBa_K1311002) and ligate to the report system(BBa_K1311007) and transform it.


iGEM-RFPFlu2photo1.jpg


The eight on the left are TALE containing groups and they are red. The only report system group(the two on the right) are white. The wild type TALE we generated is expressed and is able to bind to its target.

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