Team:Northeastern Boston/Workflow

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WORKFLOW

July 13th thru July 19th

We’ve begun by making agar plates, competent TOP10 E. coli, and stock antibiotics. We’ve also transformed the 4 strains for the Interlab and miniprepped the subsequent plasmids.

We’ve begun to PCR the parts for the two planned expression plasmids above (Benchling). Each part contains primers with homology arms that will match the neighboring pieces. One plasmid is for the nucleus and the other is for the chloroplast. The plan is produce a fluorescent protein in the nucleus, and a luminescent protein in the chloroplast. Both these "test plasmids," will them validate the expression cassettes for heterologous protein production.

July 20th thru July 26th

Interlab plasmids were digested with their respective enzymes and then purified from the gel—after staining with ethidium bromide and UV illumination—by column purification. The three Anderson promoters were mixed and ligated with the GFP from I13504. These three ligation mixtures, and the controls reconstituted, were used to transform by heat shock, 5 strains of TOP10 E. coli. These strains were then plated on agar plates with Cam and, after overnight incubation at 37 degrees, subcultured into liquid broth with Cam. Resulting colonies were screened with a fluorescent microscope and colonies expressing GFP were subcultured into a 96 well plate (3x3: biologicalxtechnical). One colony from each group was subcultured overnight, then miniprepped, digested, and run thru a gel to check for bands at the appropriate sizes. Strains in the 96 well plate were grown overnight, and then read for OD600 on a multiplate reader. A dilution curve was created and the wells were diluted to within 5% of 0.5. Fluorescence was then read (485/528nm).

PCR continues for the two protein expression plasmids. Some parts have been forthcoming, while others have taken repeated attempt. Template DNA is coming from a variety of sources: ChlamyCollection.org, iGEM registry, and Mayfield. The algae from ChlamyCollection, a nit-knockout strain, have been growing on TAP plates and in TAP liquid media for five days, with no signs of growth.

July 27th thru August 3rd

So far, we have the PCR’ed Gibson-ready parts Lux, psb 5’ UTR thru 16s-atpA, and pPsaD for the chloroplast protein expression plasmid. We have been unable to clone RbcL 3’ UTR or psbA 3’UTR thru psbH 3’ UTR yet.