Team:IIT Kharagpur/Results

IGEM-IIT Kharagpur- Part

Overview

In an attempt to synthesize two constructs, one for the Silk and the other for the FRET, we managed to move only in the direction of the FRET construct. Owing to time restrictions along with unsatisfactory results, the silk construct could not be worked upon. For the FRET construct, we managed to construct the part in two halves. One of the parts, BBa_K2163000, comprises of Cyan Fluorescent Protein, HIV Protease Cleavage Site, and Yellow Fluorescent Protein. This composite part was prepared by standard assembly. The other half of the FRET construct consists of pBAD/AraC promoter, RBS, and the OmpA protein. We isolated this part from an existing construct using PCR amplification. However, we failed to insert the amplified product into a backbone and thus could not complete the entire part.

RESULTS

CFP+HIV Cleavage Site:

The part was synthesized and checked using 1% agarose gel. Ligations were done in 2 sets, with the ratios 1:3 and 1:5, and 14 colonies from each were screened. A significant number showed the necessary shift.








Further, we digested the product with XbaI and PstI. We got the expected results after an agarose gel run.








CFP+HIV Cleavage Site+YFP:

The part was synthesized and checked using 1% agarose gel. Ligations were done in 2 sets, with the ratios 1:3 and 1:5, and 14 colonies from each were screened.








Further, we digested the product with XbaI and PstI. We got the expected results after an agarose gel run.






Promoter + RBS + OmpA:

We amplified the part by design suitable primers for BBa_K43013, the protease construct designed by Team Chile. We were able to standardize the PCR conditions, but failed at insertion into a backbone.








Further Possibilities:

Through this year’s project, we realised the potential of the FRET system. And accordingly, we will continue to work on the same, and try to find application in various areas. We were disheartened that we could not crack the Silk construct. Although, we will continue work on this construct too, and hope to complete the same. The silk construct require more modification to enhance efficiency. We plan to tackle the problem as we go along.