Team:IONIS-PARIS/labwork/Experimental 2.html
Experimental Results
Part amplification
Complete PCR protocol: Click here
The BBa_K2282006 part, inducing constitutive amilCP+DSbox expression, has been amplified by PCR using primers specific to Prefix and Suffix BioBricks. The quantity obtained was estimated based on the electrophoresis results.
→ We successfully amplified our second composite part. The bands obtained correspond to the expected size of 925 bp (part without VF2 and VR binding sites).
Part BBa_K2282006 estimated concentration after PCR clean-up: 10,8 ng/µL
Digestion and ligation
Complete digestion protocol: Click here
The pSB1C3-BBa_J04450 plasmid, taken from the iGEM distribution kit, was amplified through bacterial transformation and purified by liquid culture and miniprep. This plasmid induces mRFP constitutive expression associated with a resistance to chloramphenicol, and was digested using EcoRI and PstI restriction enzymes. The quantity of pSB1C3 backbone obtained was estimated based on the following electrophoresis results. We also digested our BBa_K2282006 part with the same restriction enzymes to allow a further ligation into pSB1C3.
→ The band at 2070 bp corresponds to the pSB1C3 backbone alone and shows that it was successfully separated from the insert
Estimated pSB1C3 concentration: 8.8 ng/µL
Complete ligation protocol: Click here
Based on the estimated quantity, we were able to ligate our BBa_K2282006 part into pSB1C3 following a 1:3 ratio to create our second recombinant plasmid.
Transformation
Complete bacterial transformation protocol: Click here
DH5-α e.coli were transformed with the pSB1C3-BBa_K2282006 ligation product, and cultivated at 37°C for 3 days on LB medium added with chloramphenicol.
→ The presence of blues colonies show that we have obtained our pSB1C3-BBa_K2282006 plasmid, inducing the constitutive amilCP+DSbox expression. The presence of red colonies was predictable as we did not purify the products of pSB1C3-BBa_J04450 digestion before the ligation step. They correspond to the bacteria transformed with the non-digested backbone. We picked the isolated blue colonies in order to purify our recombinant plasmid through liquid culture and miniprep. After having successfully verified its sequence, the final plasmid was submitted to the iGEM part registry.
