Team:Northeastern Boston/Workflow
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. Bba_K1547005 is being used for pPsaD, and Bba_K1547001 is being used for tPsaD. 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 2nd
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. We are also halfway thru the nuclear expression plasmid, with the Nitrate promoter, TagBFP, Ble, and the backbone amplifying while the rest (tRBCS2, tPsaD, and pPsaD) have not. The PCR's and gels continue.
There is still no sign of growth on the nit-knockout algae plates or erlenmeyer flask. The TAP media contains ammonium, so it's concerning that the Nit- algae strain is unable to grow, since they should, according to literature, be able to. It is also unclear whether we will be able to use a gene-gun for the chloroplast transformation.
August 3rd thru August 9th
We've decided to hold efforts on the chloroplast plasmid, focusing exclusively on the nuclear-bound plasmid for now. There in no clear indication that we'll have access to a gene-gun and the literature suggests that transformation recovery periods are much greater for particle-bombarded microalgae relative to glass bead transformed microalgae. Furthermore, the Nit-knockout algae strain has exhibited no signs of growth over the past week and half. Therefore, we've redesigned the nuclear plasmid (as shown beneath). The Nit-knockout strain was dependent on an absence of reduced nitrogen in the media to drive expression. Given the slow or non-existent growth of that strain, we've ordered a Nit-competent strain thru ChlamyCollection and redesigned the plasmid as follows. It features a constitutive promoter, pPsaD, rather than an inducible promoter, Nit. Additionally, HSP70A is being used as the promoter for Zeocin resistance and will be cloned from Bba_K640001 from the iGEM repository.
