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Overview Sensitivity Tuner --- Characterisation --- Modelling Colour Generators --- Carotenoids (Orange/Red) --- Melanin (Brown) --- Violacein (Purple/Green) The Future Safety

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Week 5

Monday

Dry Work

Violacein Synthesis

Used GeneDesigner from DNA2.0 to plan what we wanted synthesised, taking into account the different genes in the operon. This would be synthesised with codon optimisation for E. coli. All forbidden restriction sites have been removed (EcoRI, XbaI, SpeI, PstI, NotI)

Wet Work

Protocol de-bugging

For the amplification project, we need to amplify pSB3K3 as a low-copy number plasmid vector in order to transfer the activator constructs into low copy number plasmid. We tried 4 different PCR reaction mixtures, and used a Zymogen PCR wash kit before running them on an 0.8% Agarose E-gel. After looking at the gel, the ladder was quite smeared and no PCR product was visible. It was found that the samples run on the gel had a very low DNA concentration. However, some PCR solution was left before the wash kit was used, and using the Nano-Drop it was determined that it had a high concentration of DNA, so the PCR worked, we just need to refine the protocols after PCR.

Amplification

Mini-prepped I746374, I746375, I746380, and I746391; incubated overnight cultures of the rest of the 11 activator constructs in preparation for miniprep.

Pigments

Melanin

Retransformed melanin plasmid into Top10, and transformed it into MG1655 to (hopefully) achieve better expression. Plated on plates optimized for pigment production, and prepared for solid-phase pigment extraction

Violacein

Retransformed violacein plasmid into Top10, and transformed it into MG1655 to (hopefully) achieve better expression.

Mixing pigments

Plated Top10 transformed with melanin plasmid and violacein plasmid and the Roche strain transformed with Duncan’s orange plasmid on the same plate, optimized for melanin production (as this is the only pigment that requires media supplementation) but without any antibiotic resistance.

Tuesday

Dry Work

Violacein gene

Confirmed that correct protein sequence was present for each gene, by using MUSCLE to compare the gene sequence given by GeneDesigner to the sequence in the NCBI database. All protein sequences were identical.

Prepared the GeneDesigner file and the ApE file of the violacein operon to be sent for sequencing.

Wet Work