CRISPR contstructs for Nanno & bacterial conjugation of plasmids into Algae

[Cameron Clarke]

Attached is a list of the vectors I sent, you should use these to transform e coli to prepare glycerol stocks (the minipreps are ~500ng/ul) (for vectors with gateway sites you will need to use a ccdb resistant strain such as db3.1). My zeocin cassettes seem to be working in other strains (CCMP537), most strains are resistant to hyg it seems(50ug/ml hyg works for CCMP1779). 

So it turns out that the common conjugation plasmids come from the MSU-PRL in Peter Wolk's lab. So I can get ahold of those. Brian is suggesting bacteria rho5 which is a derivative of sm10, using these strains it would just be necessary to add an oriT to the plasmid to be transferred. A downside to using rho5 is that is a 2,6-Diaminopimelic acid auxotroph, and that stuff is kind of expensive. The diatom paper suggests using a CEN6/ARS for an origin in yeast and the algae, I am looking to see if anyone has this sequence (it's 500 bp so it can be synthesized as one gblock). So I would redesign pNOC-stacked to use these features but I would wait on a final design until I test the cleavage of the different 2A constructs (I've got a lot of colonies now I need western blot them).

I also sent the crispr construct, have fun. To clone with it, order a pair of primers of the target that are complementary to each other but produce overhangs complementary to the overhangs of the vector when cut with BspQI. When you get the oligos, resuspend to 100 um in TE, combine 0.5 ul of each primer in 10 ul ligase mixture, add 1 ul PNK, place in 37 C for 30-60 min. Add 50 mM NaCl and heat at 95-100 C for 10 min, let the mixture cool for 1 hour (for annealing), dilute primer mix 10X. Cut the construct with BspQI, pcr cleanup. Combine 70 ng cut crispr and 1 ul of the diluted primer mix for an overnight ligation. After transformation screen with XhoI, StuI sites. Good luck, I'll let you know if the construct works for me in the current colonies I have.