Harvard BioDesign/23 July 2014

From 2014.igem.org

Sequencing results were not good The plasmid did not have the insert

Transformed Gibson product (pBbE1a- CsgA- Spy Tag) into Mach I cells ( attempt 2) Tried varying forms of Gibson product 2 transformations add 2 microliters of Gibson product (1 & 3) 2 transformations adding 4 microliters of Gibson product (2 & 4) Used different transformation tubes → green tops Transformation 1 was probably not good because I was not on the correct setting Transformations 2 and 3 arc’ed first Tried to send pulse again for 2 ---> a good read came up on the equipment Tried to send pulse again for 3----> arc’ed once again Transformation 4 went through without arcing

Grew up another colony from the transformed Mach I cell plates Grew very slowly and left to grow over night

Designed primers for opening up the pBbE1a backbone for insertion of coil- coil proteins Coil parts have homology with the flag tag?

Received sequencing results → Results looked good