Literature review and project development was the main goal. Read articles and consulted previous iGEM projects on bacterial vs. fungal laccases. Created a list of potential bacterial laccase candidates based on other uses in wastewater pollutant degradation. Began weekly graduate advisory board meetings for discussion. Checked the stability of current lab reagents and created new LB Broth, 1x TAE buffer, and Ampicillin LB Agar plates. Settled on a list of four different laccases to utilize: Bacillus subtilis, Bacillus licheniformis, Kocuria palustris, Paenibacillus glucanolyticus.

Initially began with B. subtilis bacteria obtained from team advisor Charles Bridges. Completed genomic DNA extraction using the Qiagen DNA extraction kit. Designed primers for PCR isolation of the CotA laccase of B. subtilis followed by gel electrophoresis checks. Obtained pET22b(+) vector with an existing Profilin insert from the UConn Heaslip laboratory. Ran digestions of the vector using EcoRI and XbaI to cut out previous Profilin insert. Attempted a PCR extension of CotA to flank laccase sequence with EcoRI and XbaI.

Created a document of NCBI sequences for each of the four bacterial laccases with EcoRI on the 5' end and XbaI on the 3' end. Two coding sequences were created: one with an NSP4 signal peptide attached to the laccase and one with only the lone laccase gene. Used the IDT codon optimization tool to optimize sequence for E. coli expression and sent to IDT for gBlock synthesis.

Attempted to remove the Profilin gene from the Pet22b plasmid by digesting with EcoRI and Xbal. Had difficulty with gel extraction and purification of digested vector. Transformations following ligation did not appear to result in well-isolated colonies. Other digestion attempts demonstrated that PCR reagents worked, but insert was of incorrect size.