Team:Groningen/Notebook/24 July 2009
From 2009.igem.org
Wet
GVP Cluster
Transporters
Below the PCR1.1, with New designed forward primer with mutation for EcoRI restricion site, and PCR2 of 24/07, PCR 2 shows a fragment of desired size (~261pb) and is excised for DNA extraction for PCR3. Our previously excised PCR1 fragment will be used as template(still containing EcoRI)and see if we can reproduce products PCR1 and make PCR1.1.
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PCR from PCR1 gelproduc failed. Now we try to do a gelextraction first for PCR.
Today we have been scavenging the departments for genomic DNA to clone our genes out of since the Colony PCR's did not work.
Metal Accumulation
Vectors
- Run the PCR products and restriction digest from Paul (GVP) on gel
Legenda:
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The gel shows that there is no product for K-H, K-M and AC, there is no difference seen for K-promoters / AC-promoters and the empty vectors (like K). The size of the Lac promoters seems to be okay. For all product a restriction digest should be done.
- Glycerol stocks:
Made for: Ecoli + pSB1AC3-
- J23100 (High)
- J23106 (Med)
- J23109 (Low)
Ecoli + pSBK3K-
- J23100 (High)
- J23106 (Med)
- J23109 (Low)
Ecoli + pSB1A2-
- R0010 (pLac) nr 1
- R0010 (pLac) nr 2
They were put in -80 freezer
- Transformation of E. coli Top10 with two pBAD inducible promoters
Use normal protocol. The following plasmids were transformed:
- pSB2K3-I0500 (pBAD1) --> with extra IPTG added to the LBA
- pSB1A2-K113009 (pBAD2) --> from plate 3, location 1M of the registry
- Negative control, MQ.
Plates were put o/n at 37dg.
- O/n culture of pSB3K3 vectors
Inoculate 2x ~10ml LB-Kan with:
- pSB3K3-pHigh, pMed, pLow
- pSB3K3
Put o/n @ 37dg.
Dry
The first part of the day was spent processing the data of yesterday's [paper]. However when we calculated the drop in concentration of As(III) outside the membrane vesicle we found that it did not drop significantly enough to make a model of the efflux rate of As(III) depending on concentration of Arsenic in the cell. However we were able to find a paper which a more likely candidate. [Alternate energy coupling of ArsB, the membrane subunit of the Ars anion-translocating ATPase.]
Jasper continued working on the RPU computations, the results of which are shown at Team:Groningen/Promoters. It looks like some weird things happened to the cultures. For example, two cultures which should be identical followed roughly the same growth curve upto a point and then one of them started behaving more erratically.
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