The Arsenic 1.1 plasmids that Cadets2Vets worked with in June and July were synthesized by GeneWiz in a PUC57 vector. Preliminary experiments were performed using these plasmids until unexpected results in July forced us to examine the DNA more closely.

We analyzed the sequences that were sent to GeneWiz for synthesis and compared them against the NCBI BLAST database and the iGEM Registry, and found that the sequences sent to GeneWiz were not the sequences that we had intended to synthesize. The regulator we used appeared to be a generic transcription factor and the GFP had amino acid substitutions that were unexpected. We do not think the mutations affected GFP expression but because it did not match the Part in the Registry, we needed to correct that gene.

Cadets2Vets redesigned the plasmids using the appropriate Parts and successfully cloned synthetic DNA into the pSB1C3 vector backbone using the BioBrick Assembly method. The ligation of the inserts into the backbone was verified using restriction enzyme digest (left).

We screened 2 clones for every transformation performed during the cloning of the Arsenic Circuit. The DNA gel on the left shows the products from inserting PcArsR or PArsRGFP into the pSB1C3 vector. The plasmids were digested with the specified restriction enzymes and we expected PcArsR to create a 569bp fragment and PArsRGFP to create a 969bp fragment. The backbone is 2070bp.

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The DNA gel on the right is from 2 clones where we attempted to ligate PcArsR and PArsRGFP into the pSB1C3 backbone. We used restriction enzymes to cut PArsR out of the vector, and found that the vector fragment is different sizes for the two clones we screened. We expected to have ~3000bp vector (2070 + 969bp), which is consistent with clone #1. Clone #2 is only ~2000bp, indicating that the PArsRGFP insert did not integrate. Clone #1 is the correct plasmid to keep.