Team:Groningen/Notebook/29 July 2009

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Latest revision as of 15:00, 3 August 2009

Wet

GVP Cluster

Transporters

GlpF Transformation of GlpF in pSB1AC3 + medium constitutive promotor, pNL29 and SmtA

The cells were incubated (30 min, on ice; 5 min 37; 5 min on ice) 800uLm LB was added The cells were incubated for 1h at 37 The cells wer plated on and incubated at 37 overnight. No colonies were seen on the GlpF-psB1AC3, psB1AC3 resticted, pNL and the negative control. Colonies were seen on the SmtA tagged and untagged plates.

Metal Accumulation

Vectors

Ligation reactions were transformed into 50 μL TOP10 cells using heatshock, 45 sec. 42 °C. Cells were left to recover for 1 h at 37 °C after adding 200 μL LB medium. Cell suspensions plated out along with a negative control (1 μL MilliQ water) and a positive control (1 μL) [http://partsregistry.org/Part:pSB1AC3 pSB1AC3]-High or [http://partsregistry.org/Part:pSB3K3 pSB3K3]-H. Samples were plated out in volumes of 50 μL and 200 μL and incubated ON at 37 °C.

• Restriction digest to ligate vectors+constitutive promoters with the RFP device and pBAD and pLac with pSB3K3/pSB1AC3

• • Isolate vector+ pBAD promoter with Sigma Plasmid isolation kit.
  • Restriction digest as following:

• • Expected sizes of wanted fragments:
    • pBAD: 1700bp
    • pLac: 200bp
    • pSB3K3-const. promoter: 3000
    • pSB1AC3-const. promoter: 2700
    • RBS-RFP: 1000bp
  • Cut bands from gel (pSB1AC3-M showed no bands at all, so nothing to cut out..)
  • DNA gel extraction with NucleoSpin Extract II
  • Elute with 15uL NE buffer.
  • DNA concentration was measured by NanoDrop, they were in the range of 35-5ng/uL.
  • Stored in the freezer (should be fridge)

Dry

Jasper made sure the graphing functionality can make use of tables on the Wiki. It now can (see the pie chart test for an example). By default it takes the first table on the page, but if you append '#myid' it should use the table with id myid.

Also, since the information on some of the other candidates to model doesn't seem to be sufficient and the lab work is still more or less a moving target anyway (and in fact ArsR and GlpF might even be "ahead" in the cloning race right now) we decided to stick to our arsenic model(s). So instead of focussing on new models we're going to improve the existing ones, have a look at the underlying technology (like the wiki table functionality mentioned above) and think about experiments.

Working to the model accumulation MT in matlab Ngu2006. Update of the literature table on the wiki, there is now a summery of the papers which are used for the metals. I want the text headers rotated, but its works only in chorme and mozzila.