Once the Cameos team generated entangled genes, the Cameos and Lab teams collaborated to select variants of entangled genes which were deemed most likely to be functional (%identity, BLASTx, BLASTp, Phyre²...). Then the Lab team optimized the HuGenesS sequences for cloning (addition of BsaI sites for Golden Gate claning and improvement of ribosome binding sites)
Here are the different steps that were performed by the Lab team during six weeks in the summer:
1) DNA fragments with the designed HuGenesS were ordered to IDT.
2) Golden Gate cloning of the DNA fragments into the pBAD24-Moclo vector.
3) Transformation of the Golden Gate reaction into E.coli DH5alpha and selection on selective LB agar medium
4) Amplification + Purification of the obtained transformants
5) Verification of the selected transformants by PCR and electrophoresis
6) Plasmid DNA purification from PCR transformants -> sent to a company for DNA sequencing
7) Characterisation(s) of the genes to determine their ability -> fluorescence measurement of bacteria by flow cytometry and antibiograms
Of the seven sequences that were ordered, we succeeded in cloning two. These two cloned biobricks/ parts were characterized by our Team this summer : stable_YFP_LOV coding for a small thermostable YFP and the HugenesS Knt-GFP predicted to confer resistance to aminoglycoside antibotics and to encode GFP.
The lab books for these two parts are below
Lab book on Stable_YFP_LOV
Lab book on knt-gfp HuGenesS
Open the PDF file Stable_LOV-YFP Lab book.
Open the PDF file Knt-GFP Lab book.