Team:Groningen/Notebook/5 August 2009

It's my birthday!!! (Michael)

So cake it is....

GVP Cluster

 * → ✅ buy a nice cake and eat it ;)
 * → isolate plasmids from overnight precultures
 * → ✅ perform a ligation between cut vector and GVP fragment
 * → ✅ transform E.coli TOP10 competent cells with ligation product

Ligation


 * 2 uL Ligase buffer
 * 1 ul T4 Ligase
 * 8 uL plasmid pSB1AC3 digested with PstI and SpeI
 * 4 uL insert GVP restricted with XbaI and PstI

Incubate:
 * 25°C 30min.
 * kept on ice for 10min.

Tranformation
 * add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.

Incubate:
 * 30 min @ ice
 * 50 sec 42°C
 * 2 min @ ice
 * add 800uL LB-medium
 * incubate for 1 h at 37°C
 * plate on LB-amp50 plates

Transporters
GlpF Restriction, ligation and transformation as noted below.

Metal Accumulation
Restriction


 * 37 30min
 * Inactivation on Gel
 * Extraction from gel

Ligation


 * Tranformation
 * add 4 uL of the - ligation mixture to 50uL competent e.coli top10 cells.

Incubate:
 * 30 min @ ice
 * 5 min 37&deg;
 * 5min @ ice
 * add 800uL LB
 * incubate for 1 h at 37&deg;
 * plate on LB-AMP plates (one glpF 04-08 on chloramphenicol


 * PCR to amplify SmtA and GST-SmtA
 * Use pGEX-3X-GST-SmtA to amplify SmtA-GST and SmtA
 * Use pET29a-SmtA to amplify SmtA
 * Program: MT (right PCR machine)
 * Globally: 10cycles touchdown from 65-50&deg;C, 25 cycles with Tm59&deg;C, elongation time of 40sec.
 * Mix
 * Phusion MM (incl polymerase) 12.5uL
 * Primer fw 1uL
 * Primer rev 1uL
 * Vector 0.5uL
 * MQ 10uL


 * Run program o/n and check on gel tomorrow.

Dry
Jasper found that he had forgotten to take the difference in volume between the cells and the solution into account yesterday (in the equation relating the concentration of As(III) in the cell with the concentration outside the cell). He redid the computations (solving the equation for As(III) in th cell and plotting it against the concentration in the solution for different values of K and Vmax for ArsB), but this made no qualitative difference (it just caused a scaling of the graphs). (And he determined that apparently Kostal2004 used almost the exact same concentration of cells as Meng2004, so the results should be relatively interchangeable.)

Also, Jasper found and fixed some errors in the reaction rates on the modelling page and ploughed through some papers to look for more data we could try to use/reproduce for/with our models (so far none was found, as we don't incorporate growth into our models at all and because a lot of data does not have very useful/clear units for our purposes).