IGEM:Virginia/2012/Notebook/Genetically engineered bacteriophage for diagnosis of whooping cough/2012/08/12

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(8/12 – 8/18)

We found too much contamination after the nanodrop. There were too many types of plasmids and therefore our preps were unusable.
Planned to transform BL21 pLyss E.coli with commercial T7 genome, our T7 genome, pET30a, and negative control (no DNA).
We took O.D of transformants to check if T7 had assembled and is lysing cells.
We also took some time to think about all that we had been doing. We needed to find out if the plasmids on the gel were actually the plasmids of interest. We planned that if PCR worked, we would re-transform and plate a larger volume. Then we would pick a single colony and proceed from there. Additionally, we would use BL21 only if we did not have to re-ligate. We would only use DH5-alpha if we did have to re- ligate. If PCR fails, we would do ligation.
After the cultures grew overnight, there was no growth for BL21 on LB/Kan. Lawn on BL21 + commercial T7 genome, and the individual colonies on BL21+77 genome. We transformed plasmids and BPP genome. First we found primers to PCR-amplify various genes of interest.
After performing gel electrophoresis, we found that the plasmids do not appear to contain the inserts. We therefore had to redo ligation and transformation. We tried unfolding and refolding the hCG-beta control protein and also tested pregnancy kits before and after.
We ran an experiment with DTT and a new pregnancy test, “New Choice.” We hoped to see the positive band appear after refolding of hCG-beta for DTT that left a faint band after unfolding. This shows that would show that our folding solution, when titrated, was successful.
We determined specifics for unfolding conditions and folding conditions for hCG- beta.
Ultimately, we were able to detect hCG with the pregnancy kit. We were able to break disulfide bonds so that the kit could not detect hCG, and then we remade the disulfide bonds so that the hCG was detectable again by the pregnancy kit. We also performed electrotransformation of RB50.