Team:Bielefeld-Germany/Labjournal/week2

From 2012.igem.org

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[https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week17 Week17]
[https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week18 Week18]
[https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week18 Week18]
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[https://2012.igem.org/Team:Bielefeld-Germany/Labjournal/week19 Week19]

Revision as of 11:06, 4 September 2012

Contents

Labjournal

Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19


Week 2 (05/07 - 05/13/12)

  • Primerdesign for isolation of laccases from genomic DNA of Xanthomonas campestris B100, E. coli BL21(DE3) and Bacillus pumilus ATCC7061. The forward primers were designed with T7 promotor-overhanging ends after prefix and the first 20 bases of the wanted gene. The reverse primers were designed with a HIS-Tag and tweo stop codons before suffix and the last 20 bases of the wanted gene without the stop codon.
  • Successful PCRs of laccase genes CopA from Xanthomonas campestris B100, CueO from E. coli BL21(DE3) with the isolated genomic DNA as template.
  • Because we want to characterise laccases from different bacteria we had to order the bacterial strains, which weren't available at the University Bielefeld, from [http://www.dsmz.de/|DSMZ].
  • [http://www.dsmz.de/catalogues/details/culture/DSM-7039.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|Thermus thermophilus HB27]
  • [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|Bacillus halodurans C-125]
  • [http://www.dsmz.de/catalogues/details/culture/DSM-40069.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|Streptomyces lavendulae REN-7]
  • [http://www.dsmz.de/catalogues/details/culture/DSM-40236.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|Streptomyces griseus IFO 13350]


  • After some empty agarose gels we finally isolated laccase gene CotA from Bacillus pumilus ATCC7061 as a PCR product. As template we used the plasmid we got from the Swiss working group.
  • Team Modeling: Looking for suitable Software and enzymkinetics to model the degradation of our substartes with the different laccases. Finding the Michaelis-Menten kinetics and matlab.

Monday May 7th

  • Team Student Academy: First transformation of [http://partsregistry.org/Part:BBa_J04450 BBa_J04450] and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 BBa_I13522] and plating on selective agar
    • Electroporation setup: U= 2,5 kV C= 25 µF and R= 400 Ω
Material Volume
E. coli KRX competent cells 50 µL
glycerol (10 %) 50 µL
plasmid 1 µL
    • Result: We got little colonies. There weren’t any green colonies and only some pale red fluorescent colonies.

Tuesday May 8th

  • Team Student Academy: Repetition of the transformation didn’t change the result. We made a liquid culture of [http://partsregistry.org/Part:BBa_J04450 BBa_J04450], but it did not fluoresce. Searching for mistakes and alternatives. Maybe competent cells are not that good and in case of RFP the lacI sensitivity could be the problem.

Wednesday May 9th

Thursday May 10th

  • Team Student Academy Testing the competent cells by transformation of pUC19. The transformation did not work that good, so that we produced new ones.

Friday May 11th

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