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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 7

Monday

Wet Work

Amplification

Colony PCR of 8 colonies picked from 391 in pSB3K3 transformants, 8 colonies picked from 374 in pSB3K3 transformants, 8 colonies picked from 384 in pSB3K3, and 1 colony picked from 381 in pSB3K3 transformants confirmed to contain an insert of the right length. Overnight cultures of each of those colonies as well. No 374 constructs were found to contain inserts of the right length, however 4 384 constructs were of the correct length. The 381 construct was confirmed once again to be the correct length.

Overnight,

• Restirction digests of pSB3K3, 380, 382, 385, and 390.
• Colony PCR of 10 new 391 in pSB3K3 transformants, another 374 in pSB3K3 transformants

MelA BioBrick

PCR done of the original MelA and the first MelA intermediate (without the second PstI site), to create the DNA for a PstI restriction digest. After PCR the DNA was run on a CYBRsafe-stained gel and then extracted with the QuiGen kit. A Restriction digest was carried out using the fastdigest buffer and PstI and left for three hours.

We also tested the Primer C again, as it didn't work last time. Three PCR's were done:

• Primer F and Primer C (same aliquot as used last time, to check it wasn't just an experimental error on my part)
• Primer F and a new aliquot of Primer C
• Primer F and revortexed and re-aliquoted primer C from the origonal vial

None of these produced any result - there is clearly an error in the primer that I ordered.

Pyocyanin *NEW*

The following biobricks were transformed into TOP10 and plated out in preparation for pyocyanin biosynthesis:

• I746910 (pSB1A2) - pBAD + B0034
• I723024 (pSB3K3) - PhzM
• I723025 (pSB1A2) - PhzS
• B0015 (pSB1AK3) - double terminator
• B0034 (pSB1A2) - RBS

Dry Work

We found the phzM and phzS pigments in the registry!

• BBa_I723024 (phzM)
• BBa_I723025 (phzS)

This means we can now start working on these pigments, connecting them to processing and logic systems.

Development of Matlab programs to automatically process plate reader data was continued.

Tuesday

Wet Work

Amplification

Colony PCR of 391 in pSB3K3 showed none had the correct insert. Ligations of 380, 382, 385, 390 into pSB3K3, transformed into Top10.

381 and 384 were tranformed into the arabinose strain in preparation for assaying using the plate reader.

Pyocyanin

Transformations of I746910, I723025 and B0034 worked, and were put in overnight culture in preparation for miniprepping.

I723024 and B0015 were transformed again into TOP10.

Dry Work

Wednesday

Wet Work

Amplification

• Overnight colony PCR of 380, 382, 385, 390 in pSB3K3.
• Moved 375, 392, 394, 395 into pSB3K3, transformed into TOP10.
• Overnight colonies of 381 in pSBK3K in arabinose strain and 384 in pSB3K3 in arabinose strain in preparation for plate reader run tomorrow, as well as the appropriate controls.

Pyocyanin

• Overnight restriction digest of (fill in tomorrow) in preparation for standard assembly tomorrow.

Dry Work

Planned standard assemblies to be completed by the end of September