Team:UC Chile/Cyano/Labook/april

April

Monthly Summary

PCR runs have been defective so we haven’t been able to obtain all the parts needed to assemble our constructs. Nonetheless, parts for C1.1 and C2.1 amplified and a Gibson assembly was attempted to join them. The assembly was incorrect according to a restriction enzyme digestion protocol.

Synechocytis are growing ok.

Synechocystis PCC6803

Attempted synechocytis DNA extraction according to protocol. There was no pellet and no DNA.

Synechocystis cultures were observed under acridine dye and turned out to be axenic. (upload photos). Cultures were reinoculated in fresh BG11.

Started a growth curve for Synechocystis (that was never finished).

PCR's

• 17 PCR’s were run. Some parts amplified correctly and some did not.

Comments: DNA from synechocystis as template is not good enough. Primers form secondary images of these structures)

Gibson Assembly

Constructs whose all parts could be obtained by PCR: C1.1 and C2.1. The Gibson assembly technique was used to build the constructs. Transformation followed.

Results: C1.1≈ 7 colonies

               C2.1≈ 5 colonies
               B1 ≈ 1 colony (bacto construct)
               sfGFP (control) ≈ 15, all red (contamination?)

A DNA extraction and a restriction enzyme protocols were done in order to verify constructs.

Cut regions: C1.1 -> psbAB-RFP

                          C 2.1 -> psbA2-RFP

Results: ?? (I believe size was wrong)