Team:Groningen/Notebook/19 August 2009

GVP Cluster

 * → ✅ Restriction for Assembly
 * → ✅ Gel purification of plasmid/insert
 * → Ligation of GVP into vector pSB3K3
 * → Transformation of E.coli TOP10 cells (kan.)
 * → ✅ Test promoter/GVP constructs (grow precultures)
 * → O.n. cultures of K3-H/M/L plasmids

Restriction for Assembly

The vector pSB3K3 containing the low constitutive promoter was cut with PstI and SpeI to create correct ends for insert of GVP biobrick BBa_I750016, which was cut with XbaI and PstI.


 * 16μL pSB3K3-L in MQ (0.8μg)
 * 2μL Fast digest buffer
 * 1μL PstI fast digest enzyme
 * 1μL SpeI fast digest enzyme

--


 * 10μL plasmid-GVP in MQ (?μg)
 * 6μL MQ (end volume of 20μL)
 * 2μL Fast digest buffer
 * 1μL PstI fast digest enzyme
 * 1μL SpeI/XbaI fast digest enzyme

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

Purification

A "Agarose Gel DNA Extraction Kit" Standard Protocol from Roche Applied Science.


 * → In step ten, 30μL MQ was added to the dry pellet and incubated at room temperature.

Concentrations



Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids BBa_J61002 with Zinc, Copper and Arsenic promoters and RFP with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".


 * From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
 * Plasmids were eluted with 30μL MQ and stored in the fridge

Restriction Control

Isolated plasmids were cut with fast-digest enzymes EcoRI and PstI to cut out promoter-RFP, and create ~2000bp and ~950bp fragments.




 * → From left to right: 1kB ladder, (3x) BBa_J61002-pZntR (1-3), (3x) BBa_J61002-pCueO (1-3), (4x) BBa_J61002-pArsR (1-4).


 * → The restriction pattern was as expected fragments of ~2000bp and ~950bp. In 4 or 5 lanes an additional band at ~3000bp is seen, and can be uncut/single cut plasmid.

From each promoter one cup with isolated plasmid was cut with fast-digest enzymes EcoRI and SpeI to cut out promoter, and create ~2900bp and ~100bp fragments. As a control, BBa_J61002-BBa_J23100 was also cut in the same way.




 * → From left to right: 1kB ladder, BBa_J61002-pZntR (2), BBa_J61002-pCueO (2), BBa_J61002-pArsR (2), BBa_J61002-BBa_J23100, 1kB ladder.


 * → Restriction fragments should be in the order pArsR (72bp) > pZntR (65bp) > pCueO (43bp) > J23100 (35bp), and the gel shows this order.

Floating under influence of Metal promotors

Transporters
HmtA sequencing results came in. 5 out of 7 sequences look nice. 2 of the HmtA vectors were bad with many N. So far it looks we have the right prefix and start of the gene but it seems it is a Cys mutant. Now we are wondering if we made this mutant or if we got a mutant by accedent (cys-mutants are usualy made for crosslinking experiments in membrane proteins.) We are asking the people who supplied us with the plasmid and we plan to sequence the source as well. And time for reverse sequencing to get the other half of the construct.

The PCR's 1 and dirty failed. PCR2 is excised from the gel for downstream processing. PCR1 is on hold for because of the Cys-mutation.

Metal Accumulation
so the PCR was redone. The DNA of MymT and fMT will be isolated tomorrow by Sven. Also SmtA-RBS-pre & fMT-RBS-pre will be amplified by PCR using the F1-RBS-ATG primer and the SmtA-rev / fMT-rev.
 * Run PCR products on gel and excise bands
 * The bands of MymT and fMT were found on gel at the expected hight, the band of SmtA could hardly be seen,

Dry
Jasper worked on the quasi-steady-state equations and found that the previously used equations for import+export are probably wrong as they simply substitute (d/dt)ArsRAs=0 in (d/dt)ArsR (for example), instead of looking at (d/dt)ArsRT. This can lead to strange situations like having "creating" arsenic out of thin air.

Since we are going to make the wiki a visual interactive invironment we have been looking at the possibilities for using Google Web Toolkit. That is going to be a bit harder than usual, but not impossible. On that note KB made a flowchart of our project in Freemind and we've begon designing the graphics in Inscape