Team:Groningen/Notebook/7 August 2009

From 2009.igem.org

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Wet

GVP Cluster

DONE isolate plasmids from overnight precultures
DONE perform a restriction on isolated plasmid (should result in 6000 and 3000bp fragments)
TODO plate on cultures for pure colony over the weekend
TODO cut pSB3K3 plasmid and GVP containing plasmid for ligation
TODO purify wanted fragments
www.sigmaaldrich.com

Plasmid Purification

Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids pSB1AC3 with high, 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

Concentration of Plasmids

Plasmid Conc. ng/μL 260/280 260/230 -20 box (michael Restriction Control
pSB3K3-H 25.0 ? ? B-1 ?
pSB3K3-M 32.6 ? ? B-2 ?
pSB3K3-L 33.9 ? ? B-3 ?
GVP (biobrick) 484.0 ? ? E-5 EcoRI/PstI
pSB1AC3-H-GVP-amp 106.6 2.00 2.44 A-4 EcoRI/PstI
pSB1AC3-M-GVP-amp 165.2 1.92 2.40 A-5 EcoRI/PstI
pSB1AC3-L-GVP-amp 155.0 1.95 2.44 A-6 EcoRI/PstI
pSB1AC3-H-GVP-amp-conc. 116.3 1.96 2.43 Not Stored EcoRI/PstI
pSB1AC3-M-GVP-amp-conc. 108.7 1.95 2.46 Not Stored ?
pSB1AC3-L-GVP-amp-conc. 302.8 1.88 2.45 Not Stored ?
pSB1AC3-H-GVP-amp/chl-conc. 257.1 1.92 2.41 A-7 EcoRI/PstI
pSB1AC3-M-GVP-amp/chl-conc. 224.7 1.94 2.37 A-8 EcoRI/PstI
pSB1AC3-L-GVP-amp/chl-conc. 166.1 1.95 2.44 A-9 EcoRI/PstI
pSB1AC3-L-GVP-amp/chl-oud 223.9 1.91 2.31 Not Stored EcoRI/PstI

GVP+Metal dependent promotors
Plasmids suspected to be positive for the metal dependent promotors in front of the GVP cluster were restricted for 1 h @ 37 °C using NotI. No enzyme was present anymore to do restriction of sample 4 1x (Paul code).
Samples were put on 1% (TBE) agarose gel with a 1 kB DNA marker. Postive signals should be seen a ~6000 & ~2000, negative (first lane after marker) at ~7000 & ~2500.

PSB1AC3-GVP-metalpromotors restricted with NotI - 7august2009.jpg

Transporters

GlpF


The GlpF PCRs done yesterday (PCR 1 and2) were run on gel (lanes 2,3). Also the restriction done yesterday in order to check the ligations are shown on the gel below. The restriction was performed with HaeII for details see the notebook of 06-08-09.

F102471 2009-08-07 10hr 33min LigationCheckandPCR12 noted.jpg

a band of 105 was expected for GlpF PCR1 which could not be seen on the above 1% agarose gel. Therefor 1uL of PCR1 (with 4 uL MQ and 1 uL 6x orange loading dye) was loaded to a 2% gel as shown below (arrow), since the amount of loaded DNA is low the band is not very dark.

F102471 2009-08-07 13hr 03min-2 GLPFPCR1 ntoed.jpg

Metal Accumulation

  • 2nd PCR to amplify SmtA and GST-SmtA
    • Use pGEX-3X-GST-SmtA to amplify SmtA-GST and SmtA
    • Use pET29a-SmtA to amplify SmtA
    • Program: MT NIENKE (Left PCR machine)
      • Globally: 10cycles touchdown from 65-50°C, 25 cycles with Tm gradient from 60-50°C (4steps), elongation time of 1:20.
    • Mix
MM NH4 21uL
Primer fw [10mM] 1uL
Primer rev [10mM] 1uL
Vector 1uL
HomeTaq pol 1uL
    • Run program o/n
    • Check on gel
      • No bands seen on gel, except for SmtA-GST nr 4 (highest Tm) which gave a band around 900bp... Which is too small for SmtA-GST...
--> The tubes where unfortunately not put in the correct position, so the Tm at the gradient step was ~45-55°C....


  • Transform E. coli with pGB68 (mymT)
    • Use pGB68 from filter resuspended in 40uL MQ.
    • As neg control use pSB1AC3 (incl ccdB), as pos control use pSB1AC3-low promoter.
    • Mix 2-4uL of vector with 50uL chemically competent cells (prepared 31 aug)
    • Transform using heat-shock @ 37°C

Vectors

Dry

We (Jasper and Annelies) looked at using figure 1 from Singh2008. So far it looks like it shows problems similar to those we encountered with figure 3A from Kostal2004. Specifically, if we plug in the constants for GlpF we determined earlier it looks like either the export rate or the concentration at which it reaches half its maximum rate should be negative... We weren't able to get Mathematica to use Minimize on our cost function, so we couldn't try fitting all four parameters at once, we should try this with figure 3A from Kostal2004 as soon as Klaas Bernd is back.


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