From 2009.igem.org

Weekend stuff

Culture check

Restriction analysis has raised suspicion on the vector that contains the inducible promotors pLac and pBAD. Cultures were grown to check this. Although not stated on the culture it is expected to be a LB culture supplemented with Chloramphenicol to discriminate between and .

• AC3? fMT pLAc shows no growth
• AC3? pBAD shows no growth
• AC3 fMT shows slight growth
• AC3? pLAC shows no growth

Also a seemingly unrelated culture was picked out of the incubator

• AC3 H1, fully grown and dying for quite a while. Probably old culture

* NOTE: to my fellow lab people, please state medium+antibiotics and date of inoculation.

All floating stuff

The saline solutions don't show any differences between the different samples, only pNL29 seems slightly higher which is more noticeable in the sampled that were spinned down.
The large column was covered with parafilm to prevent air particles to enter and to improve lab hygiene. Density of culture in the column appears to be higher closer to the bottom, but culture is still separated over the entire height of the column. No pictures were taken.

Metal accumulation

Pure cultures inoculated 28august2009 on LB+Amp₁₂₀ of:

• pSB1AC3 fMT #6
• pSB1AC3 pLac
• pSB1AC3 pBAD
• pSB1AC3 SmtA #3
• pSB1AC3 pLow-fMT #3
• pSB1AC3 pLow-fMT #4
• pSB1AC3 pLac-fMT #2

All yielded single colonies, which were put to ON cultures for glycerol stocks
Due to the uncertainty of the vectors of inducible promotor mentioned cultures will be grown in Chloramphenicol supplemented LB in addition to a culture in LB+Amp₁₂₀, this to reduce the necessity of restriction analysis. If it is in pSB1A2, it should not grow on Chloramphenicol.

Transformation

ON ligation of SmtA with pLac or pLow were transformed into TOP10 E. coli, 5 μL in 50 μL aliquots. Cells were incubated on ice for ~30 min. and heat shock was applied 42 °C, 45 sec. Cells were left to recover on ice prior to adding 200 μL LB followed by 1 h of recovery @ 37 °C. Transformations were plated out on LB+Amp₁₀₀ plates (50 μL & 200 μL) and incubated @ 37 °C ON.