From 2012.igem.org

Revision as of 16:43, 9 August 2012

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 and 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

For most of the ideas little information was available. For example spontaneous combustion of hay bales is probably a combination of the metabolisms of different microorganism and fungus. 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 polymerisation of lignin or pigments.

Labjournal

Please join us on our trip through the world(lab) of cultivating, cloning, PCRs, activity tests, and of course biobricking ;) We will always keep you up to date during those months.

Week1 Week2 Week3 Week4 Week5 Week6 Week7 Week8 Week9 Week10 Week11 Week12 Week13 Week14 Week15

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(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.
• Sending requests to working groups for different plasmids , which have already worked with laccases and described them in their papers. Unfortunately just one answer came back (thanks a lot to them).So we got 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.

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 isolated genomic DNA as template.

• Because we want to characterise laccases from different bacteria we had to order the bacterial strains, which weren't available at the University Bielefeld, from [http://www.dsmz.de/|DSMZ].

• [http://www.dsmz.de/catalogues/details/culture/DSM-7039.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|Thermus thermophilus HB27]

• [http://www.dsmz.de/catalogues/details/culture/DSM-18197.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|Bacillus halodurans C-125]

• [http://www.dsmz.de/catalogues/details/culture/DSM-40069.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|Streptomyces lavendulae REN-7]

• [http://www.dsmz.de/catalogues/details/culture/DSM-40236.html?tx_dsmzresources_pi5%5BreturnPid%5D=304|Streptomyces griseus IFO 13350]

• After some empty agarose gels we finally isolated laccase gene CotA from Bacillus pumilus ATCC7061 as a PCR product. As template we used the plasmid we got from the Swiss working group.
• Team Modeling: Looking for suitable Software and enzymkinetics to model the degradation of our substartes with the different laccases. Finding the Michaelis-Menten kinetics and matlab.

Week 3 (05/14 - 05/20/12)

• Cloning of CueO (E. coli), CopA (Xanthomonas campestris) and CotA (Bacillus pumilus) in pSB1C3-backbone.
• Primerdesign for isolation of laccases from genomic DNA of Bacillus halodurans C-125, Streptomyces lavendulae REN-7, Streptomyces griseus IFO 13350 and Thermus thermophilus HB27. 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.

The problem with the the Streptomyces strains is that we don't know the exact DNA sequences of the laccase genes. The strains which are available at DSMZ are not the same strains. So we have to use the sequences of relative Streptomycetes, which are available at NCBI database. So it's gonna be exciting if the designed primers bind and we will get PCR product.

Tuesday, May 15th:

• Team Activity Tests: For some pre test and characterization for our future laccase activity standard we ordered [http://www.sigmaaldrich.com/catalog/product/sigma/53739?lang=de&region=DE 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 [http://www.sigmaaldrich.com/catalog/product/sigma/a1888?lang=de&region=DE ABTS] is a well working substrate to characterize oxidizing enzym activity. So we ordered.

Thursday, May 17th:

• Team Modeling: Meeting Mrs. Lutter, a mathematics prof. of our course of studies and looking for our first model of a metabolic pathway, finding out, that we don't need such a complex model. Start thinking that we want and what we need.

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.
• Team Modeling: Our aims for modeling:

• • model the expression of the laccases in the organisms.
  • model the aktivity of our enzyms.
  • model the interesing parts of a clarification plant (the part witch are interesing for our cleaner.

Week 5 (05/28 - 06/03/12)

• Cloning of CotA (Bacillus halodurans) and Tth-laccase (Thermus thermophilus) in pSB1C3-backbone.

Tuesday, May 29th:

• Team Activity Tests: Our ordered laccase and ABTS arrived. We couldn't 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. Since we want to express the laccase in E.coli we designed the primers like before for the bacterial laccases with T7 promotor and HIS-tag.

• Team Modeling: becomeing acquainted with matlab while reading the manual

Tuesday, June 5th:

• 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)

• Team Sequencing: Sequencing of the first finished biobricks.
• Team Modeling: programming our first differential equation and finding the ODE15s function witch solves these equations.

Wednesday, June 13th:

• 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)

• Team Sequencing: Sequencing of the next finished biobricks.
• Team Modeling: Finding out, that the "normal" Michaelis-Menten kinetic isn't the right kinetic to model our situation, because therefor you need a high and stady state çoncentration of the subtrates. We have low concentrations and not really stady state. We found a transformed equation.

Thursday, June 21th:

• 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).

Friday,June 22th:

• Team Cultivation/Purification: Starting our first cultivation Experiemnts with our first Biobricks. Transformes Cell are cultivated over night for our first preculture. After finishing our work in the laboratory we decided to collect some information for the best cultivation conditions. This night we find an interessting report of the Deutsche Bundesstiftung Umwelt (DBU) with interessting fact about different Laccases. With this Report we decided to screen different forms of th Erlenmeyer flasks.

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

Monday, June 25th:

• Team Sequencing: Sequencing of the next finished biobricks.

Tuesday, June 26th:

• 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 [http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi efp-Browser] we found out that the [http://www.ncbi.nlm.nih.gov/protein/AAM77221.1 laccase] in A. thaliana is only expressed in the developing seeds. So we now have to wait for the siliques 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)

Monday, July 16th:

• 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.
• Second cultivation
• Team Modeling: We need a contact to a clarification plant to get information about clarification plant itself and perhaps to proof our cleaner with real probes. therefor we are calling Mr. ???? form the clarification plant Schloß Holte (near Bielefeld) and he invited us to present our projekt.

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

• 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.

Tuesday, July 24th:

• 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, Xanthomonas campestris pv. campestris B100 and E. coli 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.

Thursday, July 26th

• Team Modeling and Team Sponsoring: meeting Mr. ??? from the clarification plant of Schloß Holte and finding a new cooperation partner. He want to give us the information we need to equate our model and design our cleaner. On top he wants to ask if the clarification plant can sponsor our projekt.

Saturday, July 28th

• Team Activity Tests: See, we are back already! Today we received different samples from Escherichia coli, Bacillus pumilus, Bacillus halodurans C-125, Thermus thermophilus, Xanthomonas campestris pv. campestris B100 and E. coli 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 activity.

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