Revision as of 12:03, 13 September 2012

Labjournal

Team bacterial laccase: Restriction of E. coli CueO, Xanthomonas campestris CopA and the B. pumilus laccase CotA and the pSB1C3 + RFP plasmid with the same restriction enzymes, ligation of the digested products and transformation in E.coli KRX cells.
- Since we have less CopA and Tth-laccase DNA, we set an PCR from the remaining PCR approach. For the reaction look PCR table

Team Activity Tests: Our ordered laccase and ABTS arrived. We couldn't wait to start, so we set up some stocks and created an ultimate plan of how to get to know our laccase better. We found out that natrium acetate buffer (100 mM / pH 5) would give an optimal environment to our enzyme. We decided to check the activity via a photometer. The one we may use here at the Cebitec is a Tecan Microplate reader. Check "protocols" for further information. If oxidized by laccase ABTS can me measured at 420 nm. After some trial and error we found out that a concentration of 0,1 U laccase and 0,1 µl ABTS in each well is perfect for visualizing the process. We added buffer to fill each well to 200 µl.

Team Student Academy: E. coli Mach1 with pMTE cp46 His received from the working group “Fermentation Engineering”, University Bielefeld. Plasmid contains genes for GFP and kanamycine resistance. We plated it and made a liquid culture at 30°C. Result: There was an intense fluorescence. We made a glycerol stock and a plasmid isolation.

Team Bacterial Laccases: We made colony PCRs on the transformations from the day before. We got product for every transformation approach so we plated the positive colonies on new plates to make plasmid isolation. So hopefully in some days we have the plasmids with the E. coli laccase, the Xanthomonas campestris laccase and the B. pumilus laccase with the inducible t7 promotor and a HIS-tag.

Team Bacterial Laccases: After plasmid isolation we cut our plasmids with NotI to see if the colony PCR was correct and our laccases are in the backbone. The agarose gel showed that for all of the different plasmids we had at least one plasmid with DNA band on the correct hight in agarose gel.

Team bacterial laccase: We send the isolated plasmids from Xanthomonas campestris CopA, B. pumilus CotA and E.coli CueO for sequencing.

Team Bacterial Laccases: PCRs of genomic DNA from B. halodurans we ordered from DSMZ. We handled the cells in the same way we did with T. thermphilus before and soluted the lyphlized cells in water and boiled them before PCR. After PCR we purificated the product. However the product amount was so low that we had to do the PCRs again.

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 ===Wednesday May 30th===
-*'''Team Bacterial Laccases:''' After plasmidisolation we cut our plasmids with NotI to see if the colony PCR was correct and our laccases are in the backbone. The agarose gel showed that for all of the different plasmids we had at least one plasmid with DNA band on the correct hight in agarose gel.
+*'''Team Bacterial Laccases:''' After plasmid isolation we cut our plasmids with NotI to see if the colony PCR was correct and our laccases are in the backbone. The agarose gel showed that for all of the different plasmids we had at least one plasmid with DNA band on the correct hight in agarose gel.
 ===Thursday May 31st===