Team:Groningen/Notebook/29 August 2009

=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 pSB1AC3 and pSB1A2.


 * 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



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.

(Taken using light coming mainly from above and behind the test tubes (a large fluorescent tube light). Lights in front of the test tubes were switched off and a black folder held on the window side (on the right) to avoid reflections. The background was dark grey.)

✅ 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.

Metal accumulation
Pure cultures inoculated 28august2009 on LB+Amp120 of: 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.
 * pSB1AC3 fMT #6
 * pSB1AC3 pLac
 * pSB1AC3 pBAD
 * pSB1AC3 SmtA #3
 * pSB1AC3 pLow-fMT #3
 * pSB1AC3 pLow-fMT #4
 * pSB1AC3 pLac-fMT #2

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.

Transporters
HmtA

PCR1.2 and PCR2 worked