Notebook

Week 1: (8-14 June)

• Primers were designed for PCR extraction of the naphthalene upper pathway of the Nah7 plasmid from Pseudomonas putida, and to remove T7-promoters from CueO, CotA and catechol 1,2-dioxygenase biobricks. We also designed primers to do overlap extension PCR to remove the scar that was created when attaching the HlyA-tag with the CueO, CotA and catechol 1,2-dioxygenase genes.

• Received the JapLac sequence from professor Kataoka.

• Stock solutions and agar plates with and without antibiotic resistance were made.

Week 2: (15-21 June)

• Primers were designed for site directed mutagenesis to eliminate the restrictions sites for improving the NoKoGen biobricks, and primers were also designed for restriction free cloning to assemble the HlyA-tag with the CueO, CotA and dioxygenase genes, without creating a scar.

• rhlA and rhlB genes from the DNA distribution kit were transformed into E.coli DH5α and plasmid preparations were made.

Week 3: (22-28 June)

• Transformations were done to insert biobricks with the enzymes CueO, CotA, catechol 1,2-dioxygenase and the HlyA-tag genes into DH5α. Frozen stock and plasmid preparations were made of these.

• Assembled the HlyA-tag with CueO, CotA and dioxygenase with 3A assembly.

• Transformed the NahR construct, dTomato and super yellow fluorescent protein 2 (SYFP2), as well as plasmid prepared the transformed DNA and evaluated with PCR followed by further evaluation by agarose gel electrophoresis.

• Designed primers for sequencing of the Nah7 pathway, as well as primers flanking the entire pathway.

• Made an attempt to extract the Nah7 plasmid using the designed primers, including gel electrophoresis of the PCR product. The gel showed that the PCR had not worked.