Team:Bielefeld-Germany/Labjournal

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We began our time in the lab with the cultivation of ''Xanthomonas campestris'' B100 and ''E. coli'' BL21(DE3) to isolate the genomic DNA to do PCRs and purify the desired laccase ORFs. In Order to do this we at first had to designed the PCR-primers. We decided, that the forward primers had to included the prefix, the T7 promotor, the RBS and the first 20 bases of the gene of interest and the reverse primers should consist of the last 20 bases of the gene of interest, a His-tag, followed by two stop codons and the suffix. Furthermore we started making preparations for the [https://2012.igem.org/Team:Bielefeld-Germany/Human_Practices/StudentAcademy Student Academy]. The Students Academy is a week-lasting summer school (9th to 13th of July) we got the chance to take part in organizing it. It is distinguished with a lot of presentations and lessons for pupils, but also guided experiments they had to do by themselves. Therefore the general idea for our experimental part was to give the students an understanding of the principle methods in biotechnology and synthetic biology by using fluorescent proteins. So the first step was to searched the Parts Registry for two plasmids with different fluorescent proteins and antibiotic resistances, respectively.  
We began our time in the lab with the cultivation of ''Xanthomonas campestris'' B100 and ''E. coli'' BL21(DE3) to isolate the genomic DNA to do PCRs and purify the desired laccase ORFs. In Order to do this we at first had to designed the PCR-primers. We decided, that the forward primers had to included the prefix, the T7 promotor, the RBS and the first 20 bases of the gene of interest and the reverse primers should consist of the last 20 bases of the gene of interest, a His-tag, followed by two stop codons and the suffix. Furthermore we started making preparations for the [https://2012.igem.org/Team:Bielefeld-Germany/Human_Practices/StudentAcademy Student Academy]. The Students Academy is a week-lasting summer school (9th to 13th of July) we got the chance to take part in organizing it. It is distinguished with a lot of presentations and lessons for pupils, but also guided experiments they had to do by themselves. Therefore the general idea for our experimental part was to give the students an understanding of the principle methods in biotechnology and synthetic biology by using fluorescent proteins. So the first step was to searched the Parts Registry for two plasmids with different fluorescent proteins and antibiotic resistances, respectively.  
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Revision as of 11:04, 21 September 2012

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. 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.

Starting the project

After we found our project idea we decided to have a get-to-know-weekend with some presentations about iGEM, important methods and ideas for human practices. We also held presentations about other possible iGEM projects to extend our horizon, as there were: e.g. RNA aptamers and magnetotactic bacteria. But the most important part of this weekend was the growing as a team. We realized that we all had one summer to work together, have fun together and most important to stand up together as a team. Now it was time to organize the work and find a suitable task for everyone. In a developing team a lot of different jobs have to be done, e.g.:

  • finding sponsors
  • communication with the public
  • human practices
  • wiki- and homepage-design
  • modelling
  • a forum for exchange of information
  • a joker, who entertains the team and lifts the mood

And finally lab work began, feel free to follow us in our weekly labjournal and have a look how our labwork, results and of course problems and their solutions, evolved.

Summary of Week 1 (04/30 - 05/06/12)

Start of our WET LAB time.


We began our time in the lab with the cultivation of Xanthomonas campestris B100 and E. coli BL21(DE3) to isolate the genomic DNA to do PCRs and purify the desired laccase ORFs. In Order to do this we at first had to designed the PCR-primers. We decided, that the forward primers had to included the prefix, the T7 promotor, the RBS and the first 20 bases of the gene of interest and the reverse primers should consist of the last 20 bases of the gene of interest, a His-tag, followed by two stop codons and the suffix. Furthermore we started making preparations for the Student Academy. The Students Academy is a week-lasting summer school (9th to 13th of July) we got the chance to take part in organizing it. It is distinguished with a lot of presentations and lessons for pupils, but also guided experiments they had to do by themselves. Therefore the general idea for our experimental part was to give the students an understanding of the principle methods in biotechnology and synthetic biology by using fluorescent proteins. So the first step was to searched the Parts Registry for two plasmids with different fluorescent proteins and antibiotic resistances, respectively.

Monday April 30th

Monday April 30th

Monday April 30th

Monday April 30th

Monday April 30th


Week 1 (04/30 - 05/06/12)

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 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 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. More information...
  • In a paper we found a research group who worked with the laccase 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 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 promoter, 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 BBa_J04450, a Plasmid with RFP and chloramphenicol resistance (but lacI and CAP sensitive), BBa_J23100, a plasmid with RFP and ampicillin resistance and 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.

Summary of Week 2 (05/07 - 05/13/12))

Finding our first sponsors.

Summary coming soon


Week 2 (05/07 - 05/13/12)

Contents


weekly seminar:

  • Found our first sponsors: Evonik, BioCircle and Merck, now treaties have to be created and signed
  • Julia is working on the database
  • Decision to organize waver sell to fill up our petty cash
  • Gabi and Isabel are designing a poster for the waver sell
  • For our human practices we wanted to find a sociology student, willing to think about bioethics, but failed
  • Our video is nearly done, is cutted and only needs be underlain with music


  • Team Modeling: Looking for suitable software and enzymkinetics to model the degradation of our substrates with the different laccases. Finding the Michaelis-Menten kinetics and matlab.

Monday May 7th

  • Team Student Academy: First transformation of BBa_J04450 and BBa_I13522 and plating on selective agar.
    • Electroporation setup: U= 2,5 kV C= 25 µF and R= 400 Ω
Material Volume
E. coli KRX competent cells 50 µL
glycerol (10 %) 50 µL
plasmid 1 µL
  • Result: We got little colonies. There weren’t any green colonies and only some pale red fluorescent colonies.

Team Bacterial Laccases:

  • More PCRs of laccase genes CopA from Xanthomonas campestris pv. campestris B100 and CueO from E. coli BL21(DE3) with the isolated genomic DNA as template and Xcc_LAC_FW_T7 / Xcc_LAC_RV_HIS and E.coli_LAC_FW_T7 / E.coli_LAC_RV_HIS primer pairs.
  • Since we wanted to characterize laccases from different bacteria we had to order the bacterial strains which weren't available at the University Bielefeld from DSMZ. Below is a list of the ordered strains and the laccases we want to isolate from this strains.
  • We ordered S. lavendulae sp. lavendulae ATCC 14158. Originally we wanted the strain Streptomyces lavendulae REN-7 but this strain isn't available at DSMZ. So we now hope that the laccase gene STSL from Streptomyces lavendulae REN-7 is similar to that from S. lavendulae sp. lavendulae ATCC 14158 because there's no DNA sequence for the laccase from this strain available.
  • We wanted the laccase EpoA from Streptomyces griseus IFO 13350. This strain was not available so we ordered Streptomyces griseus ATCC 10137. Unfortunately for this strain are no blast results after blasting the laccase from Streptomyces griseus IFO 13350 against database. So we decided to make primers for the laccase sequence from Streptomyces griseus IFO 13350 in the hope that the sequences are similar enough to get a PCR product.

Tuesday May 8th

  • Team Student Academy: Repetition of the transformation didn’t change the result. We made a liquid culture of BBa_J04450, but it did not fluoresce. Searching for mistakes and alternatives. Maybe competent cells are not that good and in case of RFP the lacI sensitivity could be the problem.
  • Team Bacterial Laccases: After some empty agarose gels we finally isolated the laccase gene CotA from Bacillus pumilus ATCC7061 as PCR product with the desired overhangig end. As template we used the plasmid we have got from the Swiss working group.

Wednesday May 9th

  • Team Activity Test: From the information we collected during our literature research we created a protocol for our first experiments. 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. Also we found out that sodium acetate buffer (100 mM / pH 5) would give an optimal environment to our enzyme. So let´s have a look at our protocol:

Initial laccase activity test:

100 mM sodium acetate buffer, pH 5.0

5 mM ABTS

8 U laccase

ad 200 µL deionized H20

Also we talked about further characterization after accomplishing the first experiments and confirming that the used concentrations are a good choice. We are planning to buy and characterize the laccase from T.versicolor (TVEL0), to have a comparison to our future recombinant laccases. That laccase we are going to analyze in sodium acetate buffers that are adjusted to pH 1, 3, 5, 7 and 9. Further we are going to analyze the effect of different temperatures on the enzymes activity. For that we will first do some more research on the temperatures of the waste water in clarification plants here in Germany. Also we found out that an addition of copper does enhance the laccases activity, so we are going to do some measurements with copper concentrations from 0.1 mM to 0.5 mM in each sample. This seems like some great experiments for the start, so next we are going to order what we need to do the measurements.

Thursday May 10th

  • Team Student Academy Testing the competent cells by transformation of pUC19. The transformation did not work that good, so that we produced new ones.
  • Team Bacterial Laccases PCR on Thermus thermophilus genomic DNA. First we dissolved some of the lyophilized powder in water and for opening the cells we boiled them for a few minutes. The primers we used were T.thermo_LAC_FW_T7 and T.thermo_LAC_RV_HIS to get the laccase with the same overhangs described in Monday April 30th. Finally with additional DMSO and GC-buffer we had a product of the GC-rich laccase.

Friday May 11th

  • Team Activity Tests: 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.


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