Team:Bielefeld-Germany/Labjournal/week1

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 sent requests for different plasmids with the desired laccase genes 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 [http://www.ncbi.nlm.nih.gov/protein/194015788 CotA] from Bacillus pumilus ATCC7061 and an ampicillin resistance from the Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biomaterials in Switzerland. They promised to send us the plasmid pBpL6. [http://www.biomedcentral.com/1472-6750/11/9 More information...] For an uniformly labeling we will further call this laccase BPUL.
• In a [http://www.ncbi.nlm.nih.gov/pubmed/21790191 publication] we found a research group who worked with the laccase [http://www.ncbi.nlm.nih.gov/protein/21230052 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 research group at our university. We will call this laccase XCCL in our wiki from now on.
• We found a [http://www.springerlink.com/content/x33205rp257397kr/ publication] 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. We decided to isolate the laccase from E. coli BL21(DE3) because this strain is available in our lab.
• Generating new competent E.coli KRX cells.
• For extraction of genomic DNA we cultivated 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) and for isolation of bpul from Bacillus pumilus ATCC7061 from the plasmid we hope to get. We decided before that we want to express the laccases under control of an inducible T7 promoter so we can choose the time point of induction. Additionally we decided that we want to use a c-terminal His-tag. Therefore the forward primers were designed with T7 promoter, RBS and the first 20 bases of the wanted gene. The reverse primers were designed with the last 20 bases of the wanted gene without the stop codon, a His-Tag and two stop codons. Primers: Xcc_LAC_FW_T7, Xcc_LAC_RV_HIS, E.coli_LAC_FW_T7, E.coli_LAC_RV_HIS, B.pumi_LAC_FW_T7 and B. pumi_LAC_RV_HIS

Tuesday May 1th

• Team Student Academy:
  • Searching for two plasmids with different fluorescent proteins 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

• Team Activity Test: Good morning everybody and welcome to the labjournal of Team Activity Tests. Today we started our work with some literature research about enzyme activity tests, laccases and its substrates. So today was filled with online research, reading papers and collecting information about the laccases our team decided to use.

Thursday May 3th

• Team Bacterial Laccases:
  • After the vector with the laccase gene bpul from Bacillus pumilus arrived, we transformed it into the competent E.coli KRX to have a larger amount of vector. The protocol we used was as followed:
    • The electroporation setup: U= 2,5kV, C= 25 µF and R= 400 Ω
    • Since we did not know the efficient of our competent KRX we used two different E.coli volumes for the transformation, 50 µL and 100 µL. We gave 50 µL 10% Glycerol to the reaction tubes with 1 µL 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

• • PCR program

Cycle between step 2 and 4 35 times.

Friday May 4th

Team Bacterial Laccases:

• We did Colony PCR on the transformed the Bacillus pumilus CotA plasmid. Unfortunately the control with colony PCR didn't work. So we just picked some colonies for plasmid isolation in the hope that on the AMP plate were no false positives colonies.

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