|Having worked out why our previous work was not going as planned, we spent the rest of this month trying to get all the rest of our parts as close to completion as possible. We were able to add BioBrick sites to the genes AraC and glnG, and to the promoters GlnAp2 and pBAD. However, because we ran out of time to get it re-synthesized, we were not able to prepare the promoter Plac/ara, which was going to drive our whole system. We did not have time to clone our pieces into a submission plasmid or run the necessary tests to confirm they work as planned.
Hopefully next year's team will be able to start up with our prepared pieces!
We set up 3ml cultures of bacteria transformed with plasmids containing pBAD, araC, glnG, GlnAp2 and Laci.
These samples were then miniprepped and digested with EcoRI and PstI.
When we ran these digestions on a gel, only glnG turned out the right size, but when we purified it we only got 4.6ng/ul.
We repeated our PCRs and digestions, this time the gels showed LacI, araC, pBAD and a GFP gene were all the correct size, so these were purified. It was determined we should use a 2.5% gel for the glnAp2 gene due to its small size, rather than using the standard 1% gel.
We attempted to ligate our purified glnG into pSB1C3, as well as araC and pBAD into pSB1C3. These ligations were then transformed overnight before 3ml cultures were prepared.
We miniprepped araC, glnG and pBAD (ligated into pSB1C3) and also lacI, pSB1C3, and GFP. We also made glycerol stocks of these parts.
However, gels showed that none of these inserts had been successful - we believe that the digestion of the plasmid backbone did not work.
Nevertheless, we also repeated a miniprep of glnG and glnAp2. Nanodrop results: glng = 302.9ng/ul glnAp2 = 116.7ng/ul
We PCRd these parts and digested them, but did not have time to prepare more plasmid backbone and ligate them in.