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September 23rd

3A Assembly

We're working further towards completion of K115030 and K115035 today by transforming the ligated K115022 and K115027 into commercial competent TOP10 (invitrogen).

Growing induced cultures

We've started growing induced 30ºC and 37ºC cultures today for more luciferase activity measurements. Cultures grown contained either pSB1AT3, K115012, K115029, K115031, K115032, K115034 and K115036 (as it showed to be a good construct in the colony PCR below). Some had different plasmids, either pSB1AT3 or pSB1AK3, so we've grown them on Ampicillin to minimize growth differences. We might have to consider putting K115012 (the only one in pSB1AK3) in pSB1AT3 to make strains more similar.

We've also lysed cultures grown yesterday, as they were already around OD=0.6. We had planned all cultures to be finished somewhere at the same day, but our shaker could not get below 26ºC therefore the strains grew faster.

Colony PCR

A colony PCR was performed on the K115032, K115033 and K115036 constructs. The results are displayed in figure 1. It was decided that 32 and 33 will be ligated and transformed again, colony A of K115036 will be used for miniprepping, transformation and future experimentation.

figure 1: Colony PCR of K115032, K115033 and K115036 transformations