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Team:Groningen/Notebook/5 August 2009
From 2009.igem.org
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| + | 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 V<sub>max</sub> for ArsB), but this made no qualitative difference (it just caused a scaling of the graphs). (And he determined that apparently [[Team:Groningen/Literature#Kostal2004|Kostal2004]] used almost the exact same concentration of cells as [[Team:Groningen/Literature#Meng2004|Meng2004]], so the results should be relatively interchangeable.) | ||
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| + | Also, Jasper found and fixed some errors in the reaction rates on the modelling page. | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} | ||
Revision as of 14:40, 5 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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It's my birthday!!! (Michael)
So cake it is....
Wet
GVP Cluster
- → DONE buy a nice cake and eat it ;)
- → TODO isolate plasmids from overnight precultures
- → DONE perform a ligation between cut vector and GVP fragment
- → DONE 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
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- 37 30min
- Inactivation on Gel
- Extraction from gel
Ligation
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- Tranformation
- add 4 uL of the - ligation mixture to 50uL competent e.coli top10 cells.
Incubate:
- 30 min @ ice
- 5 min 37°
- 5min @ ice
- add 800uL LB
- incubate for 1 h at 37°
- plate on LB-AMP plates (one glpF 04-08 on chlorophenacol
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.
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