Part:BBa_K748002:Experience

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Applications of BBa_K748002

The iGEM TecCEM Collegiate team decided to further characterise this biobrick. The biobrick, located in the Plate 1-3N well of the 2014 iGEM Distribution Kit, was resuspended by using 10 μl of dH2O and transformed into E. coli TOP10 chemocompetent cells for plasmid (pSB1C3) production driven by chloramphenicol (CAM) resistance. Transformed colonies were selected for biomass production, and then plasmid extraction by miniprep was carried out. After verifying the plasmid presence with electrophoresis, a restriction with XbaI and PstI was performed. The biobrick was recovered by agarose gel electrophoresis and Pure Link (Sigma-Aldrich) purification. Then, ligation was performed with the pSB1C3 backbone containing J23101 promoter, which was previously restricted with SpeI and PstI. It was transformed into DH5 E. coli strain, and growth overnight at 37ºC with CAM at 35 mg/mL. In order to perform an SDS-PAGE analysis, biomass production was promoted by inoculating into LB liquid medium for protein extraction. Once the culture reached approximately 1 O.D., the extraction protocol was followed: 1. Centrifuge 2 minutes/5000 rpm for pellet formation 2. Resuspension of pellet in appropriate volume of PBS. 3. Polytron treatment: 6 cycles of 30 seconds of Polytron (19,000 rpm) with 1 minute on each between each. This extraction was then loaded in a 12% SDS-PAGE gel using a Bio-Rad Precision Plus ProteinTM All Blue Standards as marker. This gel was stained with Coomassie solution ( 0.1% Coomassie Brilliant Blue R-250, 50% methanol, 10% glacial acetic acid), and unstained with appropriate solution (40% ethanol and 10% glacial acetic acid). Furthermore, a sensitivy antibiogram analysis was carried out with the protein extract at several volumes with a S. aureus culture to determine the functionality of the protein. This was done in a Petri dish with LB+CAM for a length of 14 hours.

User Reviews

The iGEM TecCEM Collegiate team worked with this part throughout the summer. Our review is that it is functional.

UNIQ44c0cf647b73033f-partinfo-00000000-QINU

UNIQ44c0cf647b73033f-partinfo-00000001-QINU