Team:Groningen/Notebook/19 August 2009

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Wet

GVP Cluster

DONE Restriction for Assembly
TODO Gel purification of plasmid/insert
TODO Ligation of GVP into vector pSB3K3
TODO Transformation of E.coli TOP10 cells (kan.)
DONE Test promoter/GVP constructs (grow precultures)

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 Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pSB3K3-L (SpeI/PstI) ? ? ? ? ?
GVP (XbaI/PstI) ? ? ? ? ?
www.sigmaaldrich.com

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.

Gel Mic 19 8 no.1.jpg Generulers 1kb marker Fermentas.jpg

→ 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.

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

Vectors

Dry

April
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July
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August
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October
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November
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