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.

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

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 http://www.jbc.org/cgi/content/abstract/M400037200v1 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. http://www.jbc.org/cgi/content/full/272/1/326?view=long&pmid=8995265 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.