Team:Groningen/Notebook/17 August 2009

GVP Cluster
E. coli TOP10 cells grown on plate with IPTG and resuspended in saline solution (0.9% NaCl). Left is control, no Gas Vesicle Proteins (GVP), Right is with IPTG induced GVP. Picture is after two days.


 * → ✅ Restriction for Assembly
 * → Gel purification of plasmid
 * → Ligation of metal promoter oligo's into vector BBa_J61002
 * → Transformation of E.coli TOP10 cells
 * → Test promoter/GVP constructs (grow precultures)
 * → Grow o.n. cultures to check ligation of pSB3K3-H/L-GVP constructs of last week (3 each)

Restriction for Assembly

The vector BBa_J61002 containing the high constitutive promoter was cut with EcoRI and SpeI to create correct ends for insert of metal promoter oligo's.


 * → Restriction in this way should cut out the original promoter, but the fragment is small and hard to detect on gel. The different size between double cut and single cut vector is to small to separate, and a bit of luck is needed to avoid self ligation of original promoter into vector.


 * 8 μL plasmid in MQ (1.0μg)
 * 8 μL MQ (end volume of 20μL)
 * 2μL Fast digest buffer
 * 1μL PstI fast digest enzyme
 * 1μL SpeI fast digest enzyme

Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.

Purification


 * → The 1% agarose MP (1xTBE) gave either a to weak, or to hard gel and new solution was made to be sure of the agarose concentration!


 * → After addition of 4μL 6x loading buffer to the restriction solutions the cups were stored on ice until the new gels were ready. The entire contents of the cups were loaded on gel to give a higher concentration of purified plasmid in the end.




 * → From left to right:1kB ladder,

Concentrations

Ligation

Tranformation

Transporters
HmtA time for a dirty experiment. Where we will do a PCR where we add PCR1 to PCR2 and add some more phusion+dNTPs. bot contain the same template. expected pcr product and primer dimer. later on the product will be cut with PstI one and EcoRI as positive control to select only the mutated HmtA ready for ligation into pSB1AC3 or any other vector cut with EcoRI and PstI.

Metal Accumulation

 * Check E. coli TOP10 transformants + pSB1AC3-mymT
 * Colony PCR on 4 different colonies (also from different ligation conc 3x and 6x)
 * Use the MymT fw & rev and the VR & VF2 primers to check the presence of mymT in the plasmid.
 * Run a program with TouchDown 10x (Tm62-50&deg;C and Elongation time 30sec) and Cycles 30x (Tm 58&deg;C).
 * Check on gel tomorrow.
 * Put o/n cultures of the colonies @ 37 warm room.


 * Make stock solutions of metals
 * 100mM Na-AsO2 (AsIII)
 * 96mM ZnCl2
 * 60mM CuSo4

Dry
Jasper worked on cleaning up the units on our modelling page. Specifically to make parameter v5 independent of the volume of cells in use. It turns out that this makes the equations quite a bit nicer. However, it also seems we did this the wrong way before on the transport page, when we looked at the results of Meng2004.