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From 2011.igem.org

Thuesday morning

• The Transformed A2-1 and A2-2 showed no colonies

• The Gel done for yesterdays PCR showed no bands

Lab Work

• A new ligation of A2-1 and A2-2 with PSB1C3 was performed and they were transformed into XL1-BLUE cells

• Digested more PSB1C3

• PCR run on the B1 colonies in the expression vector

• Gel run on the PCR product from B1 colonies

• Plasmid prep was done on the overnight cultures

• We made a digestion of the Plasmid prep with EcoRI and PstI and ran it on gel, which showed that we hadn't been able to get our cyp into the vector.

• Because the ligation won't be a succes we try to make digestions on PSB1C3 and expression vector over night, to be sure that didestions are completed.

Other Work

Damian requested the sequence for the RBS we are using. We sent him the sequence of BBa_J04500 (which has the RBS: BBa_B0034).

Since we dont know which plant CYP we will be able to express (if any...) we asked Damian, if he and his team could generate a regulating system that targets two enzymes at the same time. The idea was that the CYPs we use are homologues and have 100 % conserved regions long enough to meet the requirements of their system. In an alignment between A1 and A2, and B1 and A2, two regions of 14 and 9 bases long, repectively, were found. We have asked Damian to use theese sequences in the RNA system.