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)

Week 6: (13-19 July)

• The first step in the restriction free cloning was done with the genes for the enzymes CueO, CotA and catechol 1,2-dioxygense with the HlyA-tag. The products were loaded on a 2% gel and extracted to be used in the next step.

• Picture 5!!! Figure of gel (enz figure 2)

• We did a new 3A assembly with the correct HlyA-tag and our enzymes CueO, CotA and catechol 1,2-dioxygenase. We tried to remove the T7-promoter from the ligations from this correct assembly. This did not work.

• The modified laccase arrived. It was transformed into DH5α and the cultures were used for plasmid preparation to extract the DNA.

• Transformed our full construct with BBa_J23101 promoter, RBS, rhlA and rhlB into DH5α cells. The characterisation with CTAB-plate test started. No halos were shown.

• Digested the naphthalene degrading pathway, ran the product on an electrophoresis gel and extracted it from the gel. Ligated the gel extract to the two different promoters and used the product to transform into DH5α.