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.

• PICTURE 2!!!

• Since the NoKoGen biobricks had already been improved, transformation were done with the improved biobricks of rhlA, rhlB, and with RBS and the BBa_J23101 promoter from the kit into DH5α. After that plasmid preparation were done. This as a preparation for building the biosurfactant construct.

Week 4: (29-5 June/July)

• A first attempt to remove the T7-promoter from the biobricks with CueO, CotA and catechol 1,2-dioxygenase was done. The products were run on a gel and this showed that the PCR was unsuccessful.

• Picture 3. of gel!!!

• A transformation on the ligations with CueO, CotA and catechol 1,2 dioxygenase with the HlyA-tag gene were transformed but with no results. The assemblies were redone, but a colony PCR indicated that the assembly was unsuccessful.

• Assembled NahR with the reporter genes received from Erik Gullberg.

• Assembling rhlA, rhlB, with RBS and the BBa_J23101 promoter.

• Assembled plasmids with chromoproteins to plasmid backbones and then transformed them into DH5α. Made bactoart using the transformed bacteria.

Week 5: (6-12 July)

• The HlyA-tag we have been working with showed to be something entirely different, due to stabbing the kit wrong. A new transformation were done with the right HlyA-tag biobrick into DH5α cells.

• Different PCR methods e.g. touchdown protocol, gradients and adding DMSO was tried to remove the T7-promoter from the CueO, CotA and catechol 1,2-dioxygenase biobricks. The results were good for CueO and CotA.

• Made liquid overnight cultures of the strains containing NahR and dTomato with different salicylate concentrations.

• Continuing to assemble the rhlA, rhlB with RBS and BBa_J23101 promoter.

• Picture 4!!! Figure of Bactoart (gallery pic)