Team:Bielefeld-Germany/Labjournal

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Prologue

Starting the Team

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, activity tests, and of course 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 KRX cells.
  • Cultiviation of Xanthomonas campestris B100 and E. coli BL21. 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.
  • Ordering different bacterial strains from DSMZ.

  • 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 colurful 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 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 tweo stop codons before suffix and the last 20 bases of the wanted gene without the stop codon.
  • 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)and Tth-laccase (Thermus thermophilus) 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)

  • Primerdesign for isolating a laccase from Arababidopsis thaliana cDNA.
  • Team Activity Tests: After testing the T. versicolor laccase under conditions that are optimal (pH 5, 25°C ) according to the literature we now started further characterization under different pHs. We analyzed the laccases behavior when working in 100 mM natrium acetate buffer at pH 1, 3, 7 an 9. Result: We agree with the literature that pH 5 and also pH 6 seem to make the laccase happy. Since not all waste waters (especially those here in Germany) are not as warm as 25°C we now wonder what our laccase might do when exposed to low temperature such as 4°C. Stay tuned.

Week 7 (06/11 - 06/17/12)

  • Sequencing of the first finished biobricks.
  • Team Activity Tests: Since our Tecan microplate reader is not able to actively cool down to 4 °C we got the chance to meet the photometer Carry. Check "protocols" for further information about her. We used the same set up with 100 mM natrium acetate buffer, 0,1 U T. versicolor laccase and 0,1 mM ABTS as before but now measured at 4°C. Our team is planning to visit a municipal sewage plant for getting some insights into the water conditions there, so we will for sure test other temperatures after having more information. Let´s hope the water there is a little warmer since laccase doesn´t seem to be totally satisfied at 4°C. I wouldn´t either.

Week 8 (06/18 - 06/24/12)

  • Sequencing of the next finished biobricks.
  • Team Activity Tests and Team Immobilization: After all this characterizing we feel so much closer to our T. versicolor laccase that its about time to make some activity test under immobilized conditions. So now we are cooperating with Team Immobilization. We have thought about many ways how to immobilize the laccase and decided to give Silica Beads the first try. Check the Immobilization Team´s protocol for further information. Our main problem was how to measure the samples with all those beads in it. Tecan will probably be confused and give us some false values due to the beads that are disturbing its laser. So we need a way to get the beads out (and thus also stop the reaction) at a very precise point of time. Centrifugation wasn´t an option because it would simply take too long and not stop the reaction exactly in the second we want. While checking the internet for solutions we found Multi-Well Membrane-Bottom Filter Plates. Those are supposed to work in a similar way then our regular plates which we used for the Tecan but furthermore those plates contain a membrane that sieve the liquids through the filter when centrifugated. Thus the beads are separated and the ABTS-Buffer solution can me analyzed at 420 nm for oxidized ABTS. The plates will need a while before they arrive here at the Cebitec, so we decided to first find out what the optimal amount of beads is and whether the beads might also bind ABTS (see labjournal Team Immobilization).

Week 9 (06/25 - 07/01/12)

  • Sequencing of the next finished biobricks.
  • Team A. thaliana laccase: The thing about plants is that they have to grow. Fortunately we got 6 beautiful 4 weeks-old wildtype plants from Patrick Treffon from the Institute of Plant Physiology and Biochemistry at Bielefeld University. With the help of the efp-Browser we found out that the laccase in A. thaliana is only expressed in the developing seeds. So we now have to wait for the pods to develop.

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.
    • lac5 from Trametes versicolor
    • lac10 from Trametes versicolor
    • lac13 from Trametes versicolor
    • lac20 from Trametes versicolor
    • lac35 from Pycnoporus cinnabarinus
  • The first primerpairs were designed with standard prefix and suffix sequence and 20 bases complementary to the start and end of the ORF sequences.
  • Additional primerpairs were designed with AarI restriction site and Kozak consensus sequence before the first 20 bases from the start of the ORF (forward primers). The reverse primers were desigend with the last 20 bases of the lacase genes and a terminator overhaning end and a AarI restriction site.

    Problem: We forgot to delete the signal peptid sequences, which are present in the fungal laccases.

    Solution: Ordering the forward primers again with the first 20 bases after the signal peptides.

Week 12 (07/16 - 07/22/12)

  • Second cultivation

Week 13 (07/23 - 07/29/12)

  • Team A. thaliana laccase: Our plants had a great time during the last weeks in the climate chamber. So today it was time for them to donate their seeds for RNA isolation, cDNA synthesis and a PCR (check protocols). We ran an additional sample with actin primers as a positive control. However both samples did not show any bands. Maybe the high salt concentration in our sample was responsible or the laccase concentration in the 1:10 deluted cDNA was too low. We will do some washing and try again.
  • Some of our team members were at CAS conference in Munich from 07/23 - 07/25/12.
  • Successful PCRs of laccase gene lac5 from Trametes versicolor with plasmid DNA from Uni Greifswald as template.
  • Team Activity Tests:Today was the moment of truth. We received the first recombinant laccases from the cultivation team. We started with testing the supernatant from the cultivated cells to check whether they secrete laccase. We added 0,1 mM ABTS in each well that was filled with supernatant from Escherichia coli, Bacillus pumilus, Bacillus halodurans C-125, XCC or KRX (negative control - cells without plasmid) and measured as usual. There was no change in the OD so that we assume that no secretion takes place. Afterwards we tested potential laccase activity by filling each well with laccase and buffer (check protocols to see which buffer we used) and added 0,1 mM ABTS. Unfortunately no activity was seen. We will try again soon.
  • Team Activity Tests: See, we are back already! Today we received different samples from Escherichia coli, Bacillus pumilus, Bacillus halodurans C-125, Thermus thermophilus, XCC and KRX (negative control - cells without plasmid). The cells were all cultivated at 30°C and supplied with copper. We tested each one of them but none of them showed laccase activiy.

Week 14 (07/30 - 08/05/12)

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