Team:Groningen/Notebook/12 August 2009

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Wet

GVP Cluster

DONE isolate plasmids from o.n. cultures
DONE run gel to check plasmid size (should be ~9000bp)
TODO analyse results of gel

Over Night Cultures

→ All cultures showed growth, but the low constitutive promoter containing cells showed higher growth compared to the medium promoter.
→ Also the E.coli cells with medium promoter were more concentrated on the bottem of the tubes, as compared to the low promoter, which showed a more homogenus solution.
www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids pSB1AC3 with medium and low constitutive promoters and GVP 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 20μL MQ and stored in the fridge

Gel

From each cup with isolated plasmid 5μL was mixed with 1μL 6x loading buffer and incubated for 10 minutes. Together with 1kB ladder all samples were loaded on a 1% agarose gel with 4μL EtBr.


→ Gel shows bands at ~8000bp and might indicate correct ligation of GVP cluster into pSB1AC3 vector with medium and low promoters. The absence of clear lines at ~9000bp is due to the uncut round shape of the plasmids.
→ The concentration of plasmids is higher for the low constitutive promoter, which might indicate more difficulty for E.coli to grow at higher GVP expression (to much energy needed).
→ Next will be restriction analysis of the plasmids!!

Concentration

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pSB1AC3-BBa_J23106-GVP (med. no.1) 125.0 1.79 2.31 ? No
pSB1AC3-BBa_J23106-GVP (med. no.2) 134.4 1.83 2.35 ? No
pSB1AC3-BBa_J23109-GVP (low no.5) 528.9 1.90 2.34 ? No
pSB1AC3-BBa_J23109-GVP (low no.5) 708.4 1.90 2.17 ? No

Restriction Analysis

Transporters

Metal Accumulation

MBP-ArsR fusion protein ligation was transformed for a second try, plated out on LB-amp100 (50 μL and concentrated after centrifugation) Positive control (pSB1AC3-high constitutive promotor) ∞ Negative control (pSB1AC3-ccdb)1 colony on the concentrated plate Ligations had no colonies.

Vectors

Dry

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