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Team:Bielefeld-Germany/Labjournal/week2
From 2012.igem.org
Contents |
Labjournal
Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18
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 RFP and GFP and plating on selective agar
- Electroporation setup: U= 2,5 kV C= 25 µF and R= 400 <math>\omega</math>
| Material | Volume |
|---|---|
| E. coli KRX competent cells | 50 µL |
| glycerol (10 %) | 50 µL |
| plasmid | 1 µL |
