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.
First project ideas were:
the detection of multiresistent pathogens
communication between bacteria ang funghi 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
Labjournal
Please join us on our trip trough the world(lab) of cultivating, cloning, PCRs, acticity tests, and of your biobricking ;) We will always keep you up to date during those months.
Week 1 (04/30 - 05/06/12)
Start of our WET LAB time.
Generating new competent E.coli cells.
Cultiviation of Xanthomonas campestris B100 and E. coli BL21, which we got from a working group at our University, and isolation of genomic DNA. The genomic DNA was needed as template for PCRs to purify the wanted laccase ORFs.
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)
Week 2 (05/07- 05/13/12)
Doing the first steps to prepare at the Student Acadamy in cooperation with the CeBiTec. Finding suitable BioBricks to develop an colourful and easy experiment. Trying to isolate the GFP and RFP containing plasmid and to transform an Plasmidmix to produce colored Agar-plates.
Primerdesign for isolation of laccases from genomic DNA of Xanthomonas campestris B100, E. coli BL21(DE3) and Bacillus pumilus ATCC7061. The primers were designed with T7 promotor-overhanging ends after prefix and HIS-Tag before suffix.
Successful PCRs of laccase genes CopA from Xanthomonas campestris B100, CueO from E. coli BL21(DE3) with the isoltated genomic DNA as template.
Successful PCR of laccase gene CotA from Bacillus pumilus ATCC7061. As template we got a vector with the laccase-ORF from the Swiss Federal Laboratories for Materials Science and Technology, Laboratory for Biomaterials in Switzerland.
Week 3 (05/14 - 05/20/12)
Cloning of CueO (''E. coli''), CopA (''Xanthomonas campestris'') and CotA (''Bacillus pumilus'') in pSB1C3-backbone.
For some pre test and characterization for our future laccase activity standard we ordered laccase from Trametes versicolor. As well we had to order a substrate that the laccase could use to demonstrate its abilities. According to the literature ABTS is a well working substrate to characterize oxidizing enzym activity. So we ordered.
Week 4 (05/21 - 05/21/12)
Successful PCRs of laccase genes from Thermus thermophilus HB27 and Bacillus halodurans C-125 with genomic DNA as template. The primers were designed with T7-Promotor-overhanging ends after prefix and HIS-Tag-overhang before suffix.
Week 5 (05/28 - 06/03/12)
Cloning of CotA (''Bacillus halodurans''), Tth-laccase (''Thermus thermophilus'') and ccdB in pSB1C3-backbone.
Team Activity Tests: Our ordered laccase and ABTS arrived. We couldnt 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.
Week 6 (06/04 - 06/12/12)
Week 7 (06/11 - 06/17/12)
Week 8 (06/18 - 06/24/12)
Week 9 (06/25 - 07/01/12)
Week 10 (07/02 - 07/08/12)
Sending a request for plasmids containing cDNA sequences of five different laccases from Trametes versicolor and Pycnoporus cinnabarinus from the Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis at Ernst-Moritz-Arndt-University in Greifswald.
Week 11 (07/09 - 07/15/12)
Ordering primers for isolation of fungal laccases from plasmids, we got from the Ernst-Moritz-Arndt-University in Greifswald. The plasmids contain the cDNA sequences of five different laccases from Trametes versicolor and Pycnoporus cinnabarinus. The first primerpairs were designed with standard prefix und suffix sequence and 20 bases complementary to the start and end of the ORF sequences.
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