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Team:Bielefeld-Germany/Labjournal/week5
From 2012.igem.org
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| - | * '''Team bacterial laccase''': | + | * '''Team bacterial laccase''': We sequenced the isolated plasmids from ''Xanthomonas campestris'' CopA, ''B. pumilus'' CotA and ''E.coli'' CueO. The results show that only the CopA sequence was ok – Our first finished biobrick. |
| + | The sequence of ''E. coli'' showed that there are 4 bases in the promotor region missing and the CotA sequence showed a mutation which lead to another aminoacid in protein sequence. | ||
===Friday June 1st=== | ===Friday June 1st=== | ||
===Saturday June 2nd=== | ===Saturday June 2nd=== | ||
===Sunday June 3rd=== | ===Sunday June 3rd=== | ||
Revision as of 10:59, 5 September 2012
Contents |
Labjournal
Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19
Week 5 (05/28 - 06/03/12)
Monday May 28th
- 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 restricted products and transformation.
- Since we have less CopA and tth-laccase DNA, we set an PCR from the remaining PCR approach. For the reaction look PCR table
Tuesday May 29th
- 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 restricted colonies. 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. pumi laccase with the inducible t7 promotor and a HIS-tag.
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 a positive controls.
Thursday May 31st
- Team bacterial laccase: We sequenced the isolated plasmids from Xanthomonas campestris CopA, B. pumilus CotA and E.coli CueO. The results show that only the CopA sequence was ok – Our first finished biobrick.
The sequence of E. coli showed that there are 4 bases in the promotor region missing and the CotA sequence showed a mutation which lead to another aminoacid in protein sequence.
