Team:Groningen/Notebook/16 September 2009

From 2009.igem.org

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Wet

GVP Cluster

Planning

TODO work out the wiki page for GVP
DONE make a doodle for presentation planning (1-19 oct.)
TODO media attention
DONE place an ethics survey link on twitter


TODO clone pArsR-GVP into pSB2K3
TODO clone repeat out of GVP cluster
TODO make glycerol stocks of constructs
TODO enter info on part registry


Plates


Name Plasmid Used Antibiotics on Plasmid No. of Colonies Date
pArsR-GVP (low) pSB2K3 Kanamycin ~25 15 sept.
pArsR-GVP (high) pSB2K3 Kanamycin ~120 15 sept.
Negative (low) x x 0 15 sept.
Negative (high) x x 0 15 sept.


→ The plates showed a low amount of colonies, and no colonies in the case of the negative plates.


→ The two plates were stored in the fridge for further use.


Cultures

The overnight cultures with LB-amp100 medium of colonies E.coli TOP10 with pLacI-GVP (no.1 and 2), pZntR-GVP (glycerol), pCueO-GVP (glycerol), GlpF (no.1 and 2), and pLacI (pSB1AC3) all showed expected growth of bacteria.


www.sigmaaldrich.com

Plasmid isolation

Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids with the "Sygma-Aldrich™ GenElute™ Plasmid Miniprep Kit".

  • From each tube 4mL of culture was collected in a 2.0mL cup (tubes from pArsR-GVP, GVP and pNL29 were combined), 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


Concentrations

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pCueO-GVP (pSB2K3) 53.3 1.90 1.88 x Yes (EcoRI/PstI)
pZntR-GVP (pSB2K3) 49.8 1.96 2.16 x Yes (EcoRI/PstI)
pLacI-GVP (pSB1A2, no.1) 526.7 1.83 2.32 x Yes (EcoRI/PstI)
pLacI-GVP (pSB1A2, no.2) 493.3 1.83 2.34 x Yes (EcoRI/PstI)
GlpF (pSB1AC3, no.1) 118.7 1.84 1.57 x Yes (EcoRI/PstI)
GlpF (pSB1AC3, no.2) 96.7 1.92 2.03 x Yes (EcoRI/PstI)
pLacI (pSB1AC3) 158.3 1.90 2.24 x Yes (EcoRI/PstI)


Restriction for Control

The plasmids from the o.n. precultures of pArsR-GVP and pSB2K3 (earlier last week) were cut with PstI and EcoRI to cut out the entire part between the pre- and suffix. The plasmids with PstI were cut with PstI for control.

Plasmid Amount μL MQ μL Fast digest buffer EcoRI fast digest enzyme XbaI fast digest enzyme SpeI fast digest enzyme PstI fast digest enzyme
pCueO-GVP 16.0 x 3.0 1.0 x x 1.0
pZntR-GVP 16.0 x 3.0 1.0 x x 1.0
pLacI-GVP no.1 2.0 14.0 3.0 1.0 x x 1.0
pLacI-GVP no.2 2.0 14.0 3.0 1.0 x x 1.0
GlpF no.1 10.0 6.0 3.0 1.0 x x 1.0
GlpF no.2 10.0 6.0 3.0 1.0 x x 1.0
pLacI (pSB1AC3) 8.0 8.0 3.0 1.0 x x 1.0


16-9 no.1.jpg Generulers 1kb marker Fermentas.jpg

→ From left to right: 1kB ladder, three samples of SJ, pCueO-GVP (pSB2K3), pZntR-GVP (pSB2K3), pLacI-GVP (pSB1A2, no.1), pLacI-GVP (pSB1A2, no.2), GlpF (pSB1AC3), pLacI (pSB1AC3)
→ The additional two lanes are a darker image of the GlpF (pSB1AC3), pLacI (pSB1AC3) lanes, to check for GlpF and pLacI insert.

Transporters

Metal Accumulation

Vectors

Dry

April
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October
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November
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