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Team:Bielefeld-Germany/Labjournal/week1
From 2012.igem.org
Contents |
Labjournal
Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15 Week16 Week17 Week18 Week19 Week20 Week21
Week 1 (04/30 - 05/06/12)
- Start of our WET LAB time.
weekly seminar:
- Do we want to order strains of Trametes versicolor and Trametes villosa?
- Gathering information about signal sequences in yeast
- Decision to create a database, so that we can easily number and inscribe our lab results
- Decision to arrange a summer school for pupils in their last year before the final exams
- Discussion about how to meet a member of the german Bundestag (the german parliament)
Monday April 30th
- Team Student Academy: We got the chance to organize one part of the first school academy “synthetic biology/ biotechnology” at the CeBiTec of University Bielefeld by arranging experiments for the pupils and by presenting us and the iGEM competition. For the experimental part our general idea was to give them an understanding of principle methods in biotechnology / synthetic biology by using fluorescent proteins. We planned the following experiments:
- Plasmid isolation of RFP/GFP from a liquid culture.
- Transformation of a plasmid mixture consisting of two different fluorescent proteins (e.g. RFP and GFP) and different antibiotic resistances into E.coli KRX. It will be plated out on LB agar plates without antibiotics and on plates containing one of the two antibiotics, which are present on the plasmids. This way we can demonstrate the effect of antibiotics as selective pressure.
- Team Bacterial Laccases: Before our lab time started we send requests for different plasmids to working groups, which have already worked with laccases we are interested in. Sadly just one working group responded to us. We got answer for a vector with the laccase-ORF CotA from Bacillus pumilus ATCC7061 from the Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biomaterials in Switzerland. They promised to send us the plasmid. So we made primers for the laccase in the same way as for E.coli and Xanthomonas campestris laccases with T7 promotor and HIS-Tag. To get the DNA sequences of more bacterial laccases, we decided to isolate them from genomic DNA over PCR.
In a paper we found the research group worked with a laccase [http://www.uniprot.org/uniprot/Q8PCX4 CopA] from Xanthomonas pv. campestris ATCC33913. Luckily the sequence of this laccase is the same in Xanthomonas campestris pv. campestris B100 which we got from a working group at our university. The same thing with a laccase from E. coli. We found papers which described the laccase [http://www.ncbi.nlm.nih.gov/protein/85674340 CueO] from E. coli W3110. After blasting this laccase we found out that E. coli BL21(DE3) has this laccase, too.
- Generating new competent E.coli KRX cells.
- Cultivation of Xanthomonas campestris B100 and E. coli BL21(DE3). The bacterial strains we got from a working group at our University. After cultivation we isolated the genomic DNA. The DNA was needed as template for PCRs to purify the wanted laccase ORFs.
- Primer design for isolation of laccases from genomic DNA of Xanthomonas campestris B100 and E. coli BL21(DE3). 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 two stop codons before suffix and the last 20 bases of the wanted gene without the stop codon.
Tuesday May 1th
- Team Student Academy: Searching for two plasmids with different fluorescent proteins behind and antibiotic resistance in parts registry. Found [http://partsregistry.org/Part:BBa_J04450 BBa_J04450], a Plasmid with RFP and chloramphenicol resistance (but lacI and CAP sensitive), [http://partsregistry.org/Part:BBa_J23100 BBa_J23100], a plasmid with RFP and ampicillin resistance and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I13522 BBa_I13522], a Plasmid with GFP and ampicillin resistance in Kit Plate 2011.
Wednesday May 2th
Thursday May 3th
- Team Bacterial Laccases:
- After the vector has arrived, we transformed it into the competent E.coli KRX which we have already made competent to have a greater amount of vector. The protocol we used was as followed:
- The electroporation setup: U= 2,5kV C= 25 µF and R= 400 <math>\omega</math>
- Since we did not know the efficient of our competent KRX we used two different E.coli volumes for the transformation, 50µL and 100µL. We gave 50µL 10% Gylcerol to the reaction tubes with 1µL of the vector DNA (Bacillus pumilus). After the transformation we plated them into ampicillin plates.
- PCR with the Xanthomonas campestris B100 and E. coli BL21(DE3) genomic DNA to isolate the laccases. Therefore we used the primers Xcc_LAC_FW_T7, Xcc_LAC_RV_HIS, E.coli_LAC_FW_T7 and E.coli_LAC_RV_HIS, which are listed under Materials.
- PCR table
- After the vector has arrived, we transformed it into the competent E.coli KRX which we have already made competent to have a greater amount of vector. The protocol we used was as followed:
| Material | Volume |
|---|---|
| Buffer (10x Phusion) | 10µL |
| Phusion Polymerase | 0,5µL |
| dNTPs | 1µL |
| Primer Mix | 1µL |
| Template DNA | 1µL |
| DMSO | 1,5µL |
| Water | 35µL |
- PCR program
| Temperature | Time |
|---|---|
| 1) 98°C | 30 sec |
| 2) 98°C | 15 sec |
| 3) 62°C | 45 sec |
| 4) 72°C | 1 min |
| 5) 72°C | 3 min |
| 6) 12°C |
Cycle between step 2 and 4 35 times.
Friday May 4th
Team Bacterial Laccases: Colony PCR on the transformed the Bacillus pumilus plasmid. Unfortunately the control with colony PCR didn't work so we just picked some colony for plasmid isolation in the hope that on the AMP plate were no false positives.
