Overview of Experimental Setup

We aimed to utilize isolated enzymes known as laccases as a potential method of combating EE2 pollution of water sources. Laccases are a prominent class of multi-copper oxidases found naturally in numerous species of fungi and bacteria. They are versatile enzymes that are proven to be capable of degrading phenolic compounds and this makes their implementation in industrial and municipal wastewater treatment attractive. We utilized NCBI to find bacterial laccase genes and IDT’s codon optimization tool to ligate the laccase into a vector and transform it into Esherichia coli. We initially wanted to use the pET-22b(+) vector (Novagen) because it contains a pelB leader sequence to allow for a system of extracellular secretion. Due to ordering difficulties during the first cycle of the project, we were unable to receive the plasmid but were donated one with an insert (Profilin gene) inserted by XBaI and EcoRI restriction enzymes by Dr.Aoife Heaslip’s lab at UConn. We aimed to remove the insert and replace it with our gblock with compatible restriction enzymes.

Summary of Results

We initially had difficulty digesting the insert and obtaining enough DNA after gel extraction of the digestion. Therefore, we struggled at first to get an initial ligation. Once our pET22b(+)ordered plasmid came in, we attempted to digest it and ligate it again. Since we had difficulty getting good DNA from the gel extractions, we attempted to use magnetic beads to separate the plasmid backbone from the part of the multiple cloning site that was removed. We evaluated inserts of several colonies with primers that annealed to our pET22(+) plasmid, with one colony that had a large enough product to be the gBlock from the Bacillus licheniformis laccase. We sent the PCR and minipreps from the plasmid to be sequenced by Eurofins and if successful, we will test if the bacterial strain can biodegrade EE2.

Figure: Colony PCR with pET-22b vector primers

• L: Ladder in first lane
• Lane 8: Sample Colony 7 demonstrates one colony which had the larger product that could be the B. licheniformis gene insert into the pET vector.