Team:Bielefeld-Germany/Test

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Summary

Beginning in January and February members of the former iGEM team from Bielefeld started seminars to inform interested students about synthetic biology, iGEM and the past Bielefeld projects. In March the final 2012 iGEM Bielefeld team was formed of 15 students and weekly meetings began. Our team was established and it was time to find a suitable project. The first weekly meeting were more like big group brainstorming and we discussed idea, which in some cases were totally different from each other. Everyone had to inform about ideas of others so that, in the end, we all could discuss together. First project ideas were:

• the detection of multiresistent pathogens
• communication between bacteria and fungi using quorum sensing
• a bacterial hand warmer
• a possibility to detect and destroy mold fungus
• something about spontaneous combustion of hay bale
• an enzyme dispenser

After some reports in media and press about the environmental effects of steroid hormones, we decided to go for hormones. From the beginning our aim was not to detect but to degrade hormones. We found several possible ways for degradation as there are the hydrolysis of estradiol-derivates with sufatases and glucoronidases. But we thought the best way to degrade steroid hormones would be with the use of laccases. Laccases have the ability to radicalize aromatic rings and can therefore be used to degrade or polymerize a broad range of substances, such as steroid hormones, special insecticides, polycyclic aromatic carbohydrates and aromatic acids. In nature laccases are often used for degradation or polymerization of lignin or pigments.

Contents 1 Monday April 30th 2 Tuesday May 1th 3 Wednesday May 2th 4 Thursday May 3th 5 Friday May 4th

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 [http://www.ncbi.nlm.nih.gov/protein/194015788 CotA] from Bacillus pumilus ATCC7061 and a 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...]

In a paper we found the research group worked with a 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 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. We decided to isolate the laccase from E. coli BL21(DE3).

• 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) and for isolation of CotA from Bacillus pumilus ATCC7061 from plasmid. The forward primers were designed with T7 promotor, RBS and the first 20 bases of the wanted gene after prefix. The reverse primers were designed with the last 20 bases of the wanted gene without the stop codon, a HIS-Tag, two stop codons and suffix sequence. 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 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

• 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 CotA from Bacillus pumilus 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% Glycerol 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

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: 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.

Woche 2

hier eine Zusammenfassung und ein read more

Woche 3

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