Team:Bielefeld-Germany/Labjournal

From 2012.igem.org

Labjournal

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. Our team was established and it was time to find a suitable project.

Find a 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

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 microorganisms 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.

Molding together to a team

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.

Find the right one for the right job

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

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

Contents

  • Start of our WET LAB time.

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 Cloning of Bacterial Laccases:

    • Before our lab time started we sent requests for different plasmids with the desired laccase genes 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 an 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... For an uniformly labeling we will further call this laccase BPUL.
    • In a publication 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 research group at our university. We will call this laccase XCCL in our wiki from now on.
    • We found a publication 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) because this strain is available in our lab.
    • We generated new competent E.coli KRX cells.
    • For the extraction of genomic DNA we cultivated 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 the isolation of laccases from genomic DNA of Xanthomonas campestris B100 and E. coli BL21(DE3) and from plasmid pBpL6 Bacillus pumilus ATCC7061.
      • In a team meeting earlier in the project we decided that we want to express the laccases under control of an inducible T7 promoter so we can choose the time point of induction. Additionally we decided that we want to use a c-terminal His-tag. Therefore the forward primers were designed with T7 promoter, RBS and the first 20 bases of the wanted gene. The reverse primers were designed with the last 20 bases of the wanted gene without the stop codon, a His-Tag and two stop codons. 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 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.
  • Team Database:
    • We decided to create a database, so that we can easily number and inscribe our lab results and that everybody has the chance to find out witch results the other groups has so far.
    • Starting to look for possibilities to design such a database.

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 Cloning of Bacterial Laccases:
    • After the vector with the laccase gene bpul from Bacillus pumilus arrived, we transformed it into the competent E. coli KRX to have a larger amount of vector. The protocol we used was as followed:
      • The electroporation setup: U = 2,5 kV, C = 25 µF and R = 400 Ω
      • Since we did not know the efficient of our competent KRX we used two different E.coli volumes for the transformation, 50 µL and 100 µL. We gave 50 µL 10% glycerol to the reaction tubes with 1 µL 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.

Friday May 4th

Team Cloning of 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

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 V. is working on the database.
  • Decision to organize a waffle sale to fill up our petty cash.
  • Gabi and Isabel are designing a poster for the waffle sale.
  • For our human practices we wanted to find a sociology student, willing to think about bioethics, but did not succeed yet.
  • Our video is nearly done, it is cut and only needs be underlain with music.

Monday May 7th

  • Team Student Academy:
    • First transformation of BBa_J04450 and BBa_I13522 and plating on selective agar. Result: We got little colonies. There weren’t any green colonies and only some pale red fluorescent colonies.
  • Team Cloning of Bacterial Laccases:
    • More PCRs of laccase genes xccl from Xanthomonas campestris pv. campestris B100 and ecol 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 screen and characterize laccases from different bacteria we had to order the bacterial strains which weren't available at Bielefeld University from DSMZ. Below is a list of the ordered strains and the laccases we want to isolate from these strains.
      • Laccase from Thermus thermophilus HB27 (look here for a publication to this laccase)
      • BH2082 from Bacillus halodurans C-125 (look here for a publication to this laccase)
      • 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. publication
      • We wanted the laccase EpoA from Streptomyces griseus IFO 13350 (for the publication look here. 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.
  • 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.

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 Cloning of Bacterial Laccases:
    • After some empty agarose gels we finally isolated the laccase gene bpul from Bacillus pumilus ATCC7061 as PCR product with the desired overhanging ends. As template we used the plasmid we 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.


  • Team Database
    • Finding a first initial design
Figure 1: The first initial design of the database.


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 Cloning of Bacterial Laccases
    • We got the ordered strains from DSMZ. So we did 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.
  • Team Database: Conceptual Design: In this phase of the design we try to find relations between the different tables and build a entity-relationship-model. This model helps to visualize the entities (our different tables) together with their attributes (the entries belonging to a table) and the relationship between these entities. The next figure shows a very easy E-R-Model.
Figure 2: E-R-Model of our database.


Summary of Week 3

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

Contents

Weekly Seminar

  • first lab service: Robert
  • our GFP, which we wanted to use for the summer school for pupils, does not work.
  • first competent cells have to be made: Julia S. and Robert.
  • Decision to buy a commercial laccase to establish the analytics and the enzyme activity tests.
  • our scientific exposé has to be translated into english: Malak
  • Last planning for our waver sell.
  • Julia S. is creating a vector for Pichia pastoris and is now searching for usable sequences.
  • The BMBF invites all german iGEM teams to Berlin to attend at the Biotechnologie2020+ strategy process.

Monday May 14th

Tuesday May 15th

  • Team Database:
    • Creating the logically design for the database. For example for our table 'Eppi' we opt for the following logic:
eppi ( ID INT(11),
DataID INT(11),
Caption TEXT,
Content TEXT,
Experiment VARCHAR(255),
Where2 VARCHAR(255) ,
Location TEXT,
Coments TEXT,
Empty ENUM ('N', 'J'),
Create_User INT(11),
Creat_Date DATETIME,
Edit_Date DATETIME,
Delete ENUM ('N', 'J')
Delete_User INT(11),
Delete_Date DATETIME )

Wednesday May 16th

  • Team Cloning of Bacterial Laccases:
  • For cloning our laccases we need pSB1C3 backbone. Therefore we we transformed BBa_J04450 (pSB1C3 with RFP) in competent KRX cells.

Thursday May 17th

  • Team Cloning of Bacterial Laccases: Plasmid isolation of Status: 500

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  • 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.

Friday May 18th

  • Team Activity Tests: Our T.versicolor laccase and the ABTS arrived! We couldn´t wait to start, so we set up the stock solutions we will need, such as sodium acetat buffer (pH 5), 10 mM ABTS and deluted laccase.

Saturday May 19th

Sunday May 20th

Summary of Week 4

Week 4 (05/21 - 05/27/12)

Contents

Weekly Seminar

  • Lab service: Isabel
  • We try to establish a collaboration with the iGEM team from SDU-Denmark.
  • Got our distribution kits
  • First successful cloning and cultivations.
  • Who wants to be a summer school teacher?
  • We will not travel to the ACHEMA because only local teams are invited.
  • Do we want to participate in the Biolympics? (It's a sports party with fun organized by the bts).


Monday May 21st

  • Team Cloning of Bacterial Laccases:
    • We wanted to clone our laccase PCR products xccl and ecol in pSB1C3 backbone. Therefore we did some restriction digests on the PCR products and the vector BBa_J04450.
  • Team Modeling: Our aims for modeling:
    • model the expression of the laccases in the organisms.
    • model the activity of our enzymes.
    • model the interesting parts of a clarification plant (the part witch are interesting for our cleaner.

Tuesday May 22nd

  • Team Cloning of Bacterial Laccases:
  • Ligation of the digested PCR products in pSB1C3 backbone and transformation in KRX electro-competent cells.

Wednesday May 23rd

  • Team Student Academy:
    • Repetition of the transformation of BBa_J04450 and BBa_I13522 with new competent E. coli KRX cells. Got intense red fluorescing colonies but no green fluorescing colonies. Made a backup of E. coli KRX BBa_J04450.
    • Asking for other plasmids containing GFP at the working groups of our University.
  • Team Cloning of Bacterial Laccases:

    • After there were no colonies on our pSB1C3 + xccl(T7)_His (Xanthomonas campestris) transformation plate we did the transformation with the same ligation preparation again. The other ligation with pSB1C3 + ecol(T7)_His (E. coli) showed colonies so we started colony PCRs to find positive colonies. Sadly the colony PCRs showed no products but the problem was that we just had the long overhang primers (E.coli_LAC_FW_T7 and E.coli_LAC_RV_HIS ). Therefore we ordered the F and R primers.

Thursday May 24th

  • Team Student Academy:
    • Made a liquid culture of E. coli KRX with BBa_J04450 at 30 °C. There was no fluorescence.
    • Transformation of BBa_J23100 into E. coli KRX. Also got intense red fluorescing colonies.
Fig. 1: Oxidation of ABTS by TVEL0 reporting the activity of TVEL0 (n=4). Measurement setup see text.
  • Team Activity Tests:
    • On our schedule today was testing our bought laccases (from now on called TVEL0) and improving our protocol in case it won't work. We got familiar with the flat bottom microplates and got a briefing into using the Tecan correctly. We started with the following setup: 100 mM sodium acetate (pH 5), 5 mM ABTS, 8 U TVEL0 laccase, ad 200 µL with deionized water. Then we measured the absorption at 420 nm every 15 seconds over a time period of 2 minutes. Immediately our samples turned dark blue but unfortunately the changeover was out of range to be detected by Tecan. With this information we needed to reduce the laccase concentration to get measurable results. We have chosen to try another measurement with 0.1 U TVEL0 laccase and it worked! The result was a nice saturation curve but it reached a too high, because not good measurable, optimum within 1 minute (Fig. 1). To avoid the loss of important data in the beginning of the reaction and to reduce the saturation OD we decided to slow everything down by using less ABTS.

Friday May 25th

  • Team Cloning of Bacterial Laccases:
  • We had to do the PCR on T. thermo laccase again because after the purification of the last PCR product the DNA amount was very low.
  • Team Student Academy:
    • Made a liquid culture of E. coli KRX with BBa_J23100 at 30 °C. Result: There was no fluorescence.
  • Team Database:
    • Yeah we have a login!!
Figure 2: Screen shot of our Login!!


Saturday May 26th

Sunday May 27th

Fig. 3: Oxidation of 0.01 mM ABTS by TVEL0 reporting the activity of TVEL0 (n=24).

  • Team Activity Test:
    • Remember our first activity measurements? The activity was awesome and reached quickly its saturation. So today we decided to have a lazy Sunday and reduce absorption maximum and reaction speed, too. For this we used 0.1 mM ABTS instead of 5 mM ABTS and we got good results. The saturation reached its maximum at a OD420 of ~1.6 after roundabout 2 minutes (see Fig. 2). Taking together we halved the maximal OD420 and doubled the reaction time until maximum is reached with using 0.1 mM ABTS instead of 5 mM ABTS.

Summary of Week 5

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

Contents

Weekly Seminar

  • We found our next sponsor: Analytik Jena AG.
  • all sub-teams have to present their results and problems next monday.
  • Things to do in the lab:Site directed mutagenisis, cultivation, purification.
  • Kevin will bring a white board for better communication in the lab.
  • Julia presented the first screenshots of her database.
  • Derya will be responsible for sequencing from now on.
  • Collaborations: Nadine will contact SDU-Denmark and Darmstadt.
  • CAS conference for Synthetic Biology (23th - 25th of july) in Munich, Miriam will read up on travel and acccommodation expenses
  • strategy process "Biotechnologie2020+" in Berlin, Gabi, Timo and Robert will represent the team
  • press release: last changes are in progress

Monday May 28th

  • Team Student Academy:
    • Made liquid cultures of E. coli KRX with BBa_J04450 and with BBa_J23100. Result: Intense red fluorescence. We made a glycerol stock and a plasmid isolation
  • Team Cloning of Bacterial Laccases:
    • Digest of ecol(T7)_HIS, xccl(T7)_His and bpul(T7)_His and BBa_J04450 (pSB1C3 + RFP) with the same restriction enzymes, ligation of the digested products and transformation in E. coli KRX cells.
    • Since we have less bpul(T7)_His and tthl(T7)_His DNA, we set an PCR from the remaining PCR approach.

Tuesday May 29th

  • Team Student Academy:
    • E. coli Mach1 with pMTE cp46 His received from the working group “Fermentation Engineering”, University Bielefeld. Plasmid contains genes for GFP and kanamycine resistance. We plated it and made a liquid culture at 37°C. Result: There was an intense fluorescence. We made a glycerol stock and a plasmid isolation.
  • Team Cloning of Bacterial Laccases:
    • We did colony PCRs on the transformations from the day before. We got product for every transformation approach so we plated the positive colonies on new plates to make plasmid isolation. So hopefully in some days we have the plasmids with the E. coli laccase, the Xanthomonas campestris laccase and the B. pumilus laccase with the inducible T7 promoter and a His-tag.
Figure 1:Saturation curves of TVEL0 activity with 0.1 mM and 0.05 mM ABTS (n=24).
  • Team Activity Tests:
    • After establishing our recipe for activity measurements we were curious about different ABTS concentrations and wanted to make sure we took the right one for our approach. With this in mind we did our activity measurement with 0.1 U TVEL0 laccase, 100 mM sodium acetate buffer (pH 5), ad 200 µl H2O dest., but with two new ABTS concentrations, namely 0.2 mM and 0.05 mM ABTS (see Fig. 1). It turned out that using 0.05 mM ABTS leads to a low maximum of saturation but reaches it quickly. With 0.2 mM ABTS the opposite occurs: the activity curve is saturated at a OD420 of ~2.7 but it needs more time to reach its maximum. Knowing this we are happy using 0.1 mM ABTS because it is saturating slowly and the maximum is not too high. 0.1 mM ABTS is therefore established!

Wednesday May 30th

  • Team Cloning Bacterial Laccases:
  • After plasmid isolation we digested our plasmids with NotI to see if the colony PCR was correct and our laccases are in the backbone. The agarose gel showed that for all of the different plasmids we had at least one plasmid with two DNA bands on the correct height in agarose gel.

Thursday May 31st

  • Team Cloning of Bacterial laccase:
    • We sent the isolated pSB1C3 plasmids with xccl(T7)_His, Bpul(T7)_HIS and Ecol(T7)_His for sequencing.

Friday June 1st

  • Team Activity Test:
    • Today we prepared for our next measurements by setting up the sodium acetate buffer in different pHs. We choose to test the activity of TVEL0 in a pH-Gradient of 1,2,5,7 and 9.

Saturday June 2nd

Sunday June 3rd

  • Team Cloning of Bacterial Laccases:
    • PCRs of genomic DNA on bhal from B. halodurans C-125 we ordered from DSMZ before. We handled the cells in the same way we did with T. thermophilus before and soluted the lyophlized cells in water and boiled them before PCR. After PCR we cleaned up the product with gel electrophoresis and PCR clean-up kit. However the DNA amount was so low that we had to do the PCRs again.

Summary of Week 6

Week 6 (06/04 - 06/10/12)

Contents

Weekly Seminar

  • As part of the sponsoring with Merck we will present our project in Darmstadt. Kevin, Nadine and Gabi travel to Darmstadt on 21th of june.

The team will gather on 14th, 15th and 18th of june to practice the presentation.

  • Participants at the CAS conference for Synthetic Biology: Hakan, Derya, Miriam, Julia S., Gabi, Malak, Timo, Robert, Isabel, Nils.
  • Robert is responsible for ordering the following BioBricks from the Partsregistry:

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  • The treaty with our sponsor BioCircle is signed.
  • All teams presented their lab results.

Monday June 4th

  • Team Cloning of Bacterial Laccases:
  • The Bacteria S. griseus and S. lavendulae has been delivered so we can start with PCRs. We set the first PCR with them as followed:
    • 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 7 mins
2) 98°C 20 sec
3) 55°C 20 sec
4) 72°C 1 min
5) 72°C 3 min
6) 12°C

Cycle between step 2 and 4 35 times.

  • Team Fungal and Plant Laccases:
    • 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.

Tuesday June 5th

  • Team Student Academy:
    • Transformation of a plasmid mixture of either pMTE cp46 His and BBa_J04450 or pMTE cp46 His and BBa_J23100. We plated both on LB agar without antibiotics and with Kanamycin. The first one was also plated on LB agar with ampicillin and the second on LB agar with chloramphenicol. Result: Works as expected. BBa_J23100 has a more intense fluorescence and was chosen for the experiment.
Figure 1:Activity measurements of the bought laccase from T.versiolor analyzed through the OD420 of oxidized ABTS in sodium acetate buffer at different pHs depending on time. Values are calculated by taking the average and standard deviation out of 4 measurements (n=4).
  • Team Cloning of Bacterial Laccases:
    • The sequencing results for isolated plasmids xccl(T7)_His, bpul(T7)_His and ecol(T7)_His came. The results showed that only the xccl(T7)_His was ok – our first finished biobrick *yeha*. We proudly name it Status: 500

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. The sequence of 'ecol(T7)_His showed that there are missing 4 bases in the promoter region and the bpul(T7)_His sequence showed a mutation which leads to another amino acid in protein sequence.

    • Again we did PCRs on T. thermophilus laccase and B. halodurans laccase with B.halo_FW_T7 / B.halo_FW_HIS and T.thermo_LAC_FW_T7 / T.thermo_LAC_RV_HIS primers and purified the product,this time with enough material for a restriction.


  • 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 sodium acetate buffer at pH 1, 3, 7 an 9. Result: We agree with the literature that pH 5 seems 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 lower temperaturers. Stay tuned.

Wednesday June 6th

  • Team Wiki: Yay for Team Wiki´s first entry. Our first steps with the iGEM Bielefeld 2012 Wiki contain thinking about contents, layouts, programming and responsibilities. Our first rules are:
    • we are programming static pages in HTML and all the other pages (those that will be updated by all team members) in wiki code.
    • we created all pages and will fill them up with some nice and beautiful content constantly from now on.
    • Our temporary banner contains our outstanding logo and a DNA but we will set up a new layout soon.
  • Team Cloning of Bacterial Laccases: Digest of tthl(T7)_His and bhal(T7)_His PCR products and ligation in pSB1C3 backbone. After that we transformed the plasmids in competent E. coli KRX cells.

Thursday June 7th

  • Team Student Academy:
    • Repeating of Transformation of 06/05 to verify the function. It is reproducible :)
  • Team Cloning of Bacterial Laccases: Colony PCRs of the transformed colonies from yesterday's transformation showed some positive PCR bands.
  • Team Modeling: becoming acquainted with matlab while reading the manual

Friday June 8th

  • Team Cloning of Bacterial Laccases:
    • We plated colonies for plasmid isolations on new plates and made a control restriction with NotI. The electrophoretic separation showed gel bands in the right height for the Tthl(T7)_His and with bhal(T7)_His.

Saturday June 9th

Sunday June 10th

Summary of Week 7

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

Contents

Weekly Seminar

  • Agatha and Saskia will perform a mRNA isolation from Arabidopsis thaliana.
  • Everything is in progress. Check.

Monday June 11th

Tuesday June 12th

  • Team Student Academy:
    • The whole experiment was tested by another team member to plan the course.

Wednesday June 13th

  • Team Cloning of Bacterial Laccases: Since the GC amount of the S. griseus and S. lavendulae laccases are high we used betain to solve the PCR problem. Addition of betain did not change anything on the results, we still didn't got our laccase DNA.
  • Team Modeling: Programming our first differential equation and finding the ODE15s function witch solves these equations.

Thursday June 14th

  • Team Cloning of Bacterial Laccases:
    • Because our PCRs have not worked well we thought it may depends on the primer annealing temperature so we did gradient PCR with the same conditions as before (PCR June 4th). But this also showed no result. Because we made Coloyn PCRs from the arrived DSMZ reaction tubes our next idea was to cultivate the bacteria in media and isolate genomic DNA.
  • 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 does not seem to be totally satisfied at 4°C. I would not either.

Friday June 15th

  • Team Cloning of Bacterial Laccases:
    • Sequencing of the pSB1C3 plasmid with bhal(T7)_His was ok. In conflict to our reference sequence there was a point mutation in the DNA sequence but this mutation doesn’t lead to another amino acid. So..next BioBrick (Status: 500

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) is ready to use!

    • The sequenced plasmid bpul(T7)_His showed again the same mutation in the laccase ORF compared to the reference sequence. We concluded that probably the PCR amplification caused the point mutation. So we did the digest of bpul(T7)_His PCR products from a new PCR, ligated it in pSB1C3 backbone and transformed it in competent KRX cells. Additionally we did the digest tthl(T7)_His and the ligation in pSB1C3 backbone again.
  • Team Fungal and Plant Laccases:
    • The whole week we were looking for different sources of the laccase sequences and the corresponding strain, we find an interessting study of the Federal Environmental Agency (Umwelt-Bundes-Amt Germany). In this study they analysed the oppurtunities to synthesis different chemical compounds with the aid of different laccase of Tramtetes versicolor. In this document we found a working group from the Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis at Ernst-Moritz-Arndt-University in Greifswald (Germany) who have isolated four sequences of different laccases of Trametes versicolor and the sequence of one Pycnoporus cinnabarinus. We have send a request for the plasmids containing cDNA sequences of five different laccases from Trametes versicolor and Pycnoporus cinnabarinus .
  • Team Database: We finished our first tabel, you can add a new entry, delete it and edit it.
Figure 1: Screen shot of our Plates table.

Saturday June 16th

Sunday June 17th

Summary of Week 8


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

Contents

Weekly Seminar

  • Julia V. presents the database to have an easier overview over the labwork, the biobricks and as a digital lab diary.
  • the last changes have been implemented into the press release, it is about time to let the world know about our project.
  • Everyone please keep the iGEM deadlines in mind.
  • Our advisor Timo presents: iGEM 101: Introduction to the wiki.
  • Julia S is presenting her progesses in designing the Pichia pastoris shuttle vector.
  • Modelling: Julia V. is thinking about how expression rates and promoter activity can be implemented into the model. Sebastian tries to model a sewage treatment plant.

Monday June 18th

  • Team Cloning of Bacterial Laccases:
    • We started Colony PCRs on the colonies from June 15th transformation and picked positive colonies to plate them for plasmid isolation. Sadly we just had positive clones for tthl(T7)_His and not for bpul(T7)_His.
  • Team Site Directed Mutagenesis:
    • Reviewed all Assembly-Standards and made a list of all illegal restriction-sites:
    • EcoRI, NotI, PstI, SpeI, XbaI (Silver), AgeI, NgoMIV (Freiburg), BamHI, BglII, XhoI (Berkeley)
    • Decided to not care for restriction-sites of the Berkeley-assembly (even if it is a great assembly for protein-fusion), because the used Vector (pSB1C3) already has two XhoI-restriction-sites
  • Team Fungal and Plant Laccases
    • We have not received any reply to our e-mail. Team meeting to discuss the further way forward. We decided to send a second wave of e-mails to the different working groups, try to call the working groups(if possible) and to look for strains of the corresponding and published sequences in additional straincollections.

Tuesday June 19th

  • Team Wiki:
    • While using the lab journal more frequently there came up some questions.
    • How detailed do we plan to write our lab journal entries?
    • Do we want to write in keywords or explain everything in full sentences?
    • Do we want to note every little detail about every successful or unsuccessful experiment or just the main important aspect?
    • We discussed, sighted some former iGEM team wikis and decided:
      • each team is responsible for their own lab journal entries
      • we divide our lab journal in weeks and days to prevent it from looking too chaotic.
      • the texts are supposed to state which team is writing, which experiment has been done and what the main aspects were. Also we will write about successful experiments, as well as problems and solutions we came up with. If possible links to protocols with further information shall be created.

Wednesday June 20th

  • Team Site Directed Mutagenesis:
    • Imported sequences of most of the used bacterial Laccases into Clonemanager and analysed their restriction-sites:
    • bhal has no illegal restriction-sites
    • ecol has one NgoMIV-restriction-site
    • bpul has one XbaI-Restriction-site and one mutation two AgeI- and two NgoMIV-restriction-sites (Decided to delete the Freiburg-restriction-sites would take too much time)
    • tthl has one PstI-restriction-site and two NgoMIV-restriction-sites (Decided to delete the Freiburg-restriction-sites would take too much time)
    • xccl has two PstI, one AgeI and seven NgoMIV-restriction-sites (Decided not to change the NgoMIV-sites, since to mutate seven would take too much time)
  • 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 steady state concentration of the substrates. We have low concentrations and not really study state. We found a transformed equation.

Thursday June 21st

  • Team Cloning of Bacterial Laccases:
    • Plasmid isolation and control digest with NotI on tthl(T7)_His and luckily this time the bands were where they should be. Again and again we did transformation of ligation with bpul(T7)_His laccase in pSB1C3 backbone..all fingers are crossed that this time we have colonies with the correct plasmid.
  • 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 lab journal Team Immobilization).
  • Team Immobilization:
    • So after a lot of reading and discussion, we decided to try immobilization using beads. Since silica beads were already available in our lab (from last year’s team), we decided to give them a try. The first challenge was to find out a convenient ratio of beads/laccase. According to the protocol of last year’s team, the ratio 1:1000 was used (1000mg beads/ 1 mg protein). Therefore we decided to try the ratios 1:500, 1:1000 and 1:1500. We prepared different buffers: HBSS buffer, Recrystallization buffer both of which were used with silica beads; in addition to Britton-Robinson Buffer, which was mentioned in publications as the best buffer for laccase immobilization. Laccases from TVEL0 were incubated with silica beads and different buffers at room temperature for 4hours on a rotator. After that, we collected the supernatants and delivered them to the team “Activity test” and waited for the good news.

Friday June 22nd

  • Team Cloning of Bacterial Laccases:
    • Because our PCR didn't work on the boiled lyophilized cells we used CASO Medium for cultivation of S. griseus and S. lavendulae.
  • Team Immobilization:
    • Unfortunately, the activity test results weren’t promising. According to publications, immobilization via covalent binding is the most widely used method. Silica dioxide bead offer only an adsorption of laccases. Therefore, we decided to order CPC-(controlled pore carrier) silica beads, to which laccases covalently bind, especially that we found some papers with protocols and activity tests proving their efficiency.

Saturday June 23rd

  • Team Cloning of Bacterial Laccases:
    • The cultured S. griseus and S. lavendulae bacterials has been centrifuged at 13.000 rpm for 5 minutes. After this step we ribolyzed the pellet in 1 ml TE-Puffer and set a PCR reaction after. But we still haven't had any results.
    • Colony PCRs on the transformations with plasmid with bpul(T7)_His and plating positive colonies.
  • Team Database:
    • In the last wekk we finished the tabels Eppi, User, Sequencing and BioBrick.
    • We have a problem with the search function, we use the false SQL statement and allready deleted entries are shown.
    • In the next week we have to write a new statement.

Sunday June 24th

  • Team Cloning of Bacterial Laccases:
    • We isolated plasmids and did control digests with NotI. We finally had a positive restriction digest for bupl(T7)_His. So we prepared this plasmids and the plasmid tthl(T7)_His which we isolated some days before for sequencing.

Summary of Week 9

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

Contents

Weekly Seminar

  • evaluation of the presentation, we did in Darmstadt as part of our sponsoring through Merck
  • Merck is strongly interested in our results, because the company has problems with lignin in the waste water (and laccases can be used for degradation of lignin, too).
  • We were advised to use silica-beads, pretreated with carbodiimide, to immoblize our enzymes
  • We decided to visit at least one sewage treatment plant as part of our Human Practices and we will try to integrate the treatment plants into our modelling.
  • Everything is prepared for our summer school. Kevin and Gabi will present the experiment and

Nadine, Kevin, Gabi, Mo, Miriam, Derya and Isabel will advise the pupils.

Monday June 25th

  • Team Cloning of Bacterial Laccases:
  • Retried the DNA isolation from S. griseus and S. lavendulae without any success.
  • Team Fungal and Plant Laccases:
    • Phonecall with the Leader of the working group of the University Greifswald Prof. Dr. U. Bornscheuer. We explained our project and asked, if we can get the sequnces of the Trametes versicolor laccases. We got the commitment for getting the laccase sequences and plasmids containing the sequences (four laccases of Trametes versicolor and one of Pycnoporus cinnabarinus).

Tuesday June 26th

  • Team Fungal and Plant Laccases:
    • 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 siliques to develop.
  • Team Shuttle Vector:
    • Prepare the YPD Media for cultivation of the yeast strains Pichia patoris X-33 (wild type) and GS115 (Invitrogen). Both organisms are provided from the chair of Fermentation Engineering (D5) from Dr. Thomas Hug.

Wednesday June 27th

Activity measurement of TVELO in sodium acetate buffer (pH5) and Briton Robinson buffer (pH 5). Measurements were taken via OD420 of oxidized ABTS.
  • Team Shuttle Vector:
    • Cultivation of Pichia pastoris X-33 and GS115 in YPD media for isolation of the genomic DNA.
  • Team Activity Tests:
    • We like our new cooperation with Team Immobilization. The thing is, that they don´t like our buffer. Sodium acetate (pH 5) seems perfect for activity tests but apparently not for immobilization. What they prefer is a Britton-Robinson buffer (pH 5). To find out whether there is a difference between the two buffers that causes different activity habits of our laccase TVEL0, we setup comparable measurements with the two buffers and TVEL0. We concluded that the laccase in sodium acetate buffer shows a slower saturation but all in all both laccase samples reach the same maximum so that it is ok for us to use both buffer systems.

Thursday June 28th

  • Team Wiki:
    • Today we browsed our wiki and were not very impressed: it's a lonesome place. So we started to think of how we could blow a little more life into it. For this, texts should appear soon on our wiki. To manage this bunch of work, we divided the subtopics of our wiki and appointed them to group members. Now everyone has a topic which he is responsible for. And that includes writing the texts, uploading pictures and keeping the represented information updated. Before anybody had the chance to disappear behind his/her notebook being busy editing his own page, we had to establish our wiki rules:
    • Use the standardized formatting as presented in our example page.
    • Try to edit your text without using HTML code as far as possible and use wiki code instead. Useful advices when using wiki code are represented on our example page, too.
    • If you want to change anything that does not belong to your scope of duties, ask kindly the person of charge and make sure he/she is fine with it.
    • Make your text more understandable by using images and charts. But remember: you are only allowed to upload pictures if you own them or if they are published without licenses.
Used primers Template successful amplification
5'AOX_FW and 5'AOX_RV pPICzA yes
3'AOX_FW and 3'AOX_RV pPICzA no
pAOX1_FW and pAOX1_RV pPICzA yes
tAOX1_FW and tAOX1_RV X-33 yes

Friday June 29th

  • Team Cloning of Bacterial Laccases:
    • Sequencing results showed that even with a new PCR product the same mutation occurs in bpul(T7)_His so it is probably already present on the plasmid which was sent to us. So we decided to use this plasmid. We give him the name Status: 500

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. Tth(T7)_His showed a positive sequencing result, too. So we have Status: 500 Content-type: text/html

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ready for use!
  • Team Shuttle Vector:
    • Primer design was done for the shuttle vector. The following primers create fragments with a 5' overlap. Were ordered the primers: pSB1C3-5aox1-f, pSB1C3-5aox1-r, 5aox1-mfalpha1-f, 5aox1-mfalpha1-r, mfalpha1-aarI-taox1-f, mfalpha1-aarI-taox1-r, taox1-phis4-f, taox1-phis4-r, phis4-kozak-his4-f, phis4-kozak-his4-r, his4-3aox1-f, his4-3aox1-r, 3aox1-pSB1C3-f , 3aox1-pSB1C3-r.

Saturday June 30th

  • Team Student Academy:
    • We prepared a script for pupils containing background information and a protocol and wrote an abstract for the school academy program.

Sunday July 1st

Summary of Week 10

Week 10 (07/02 - 07/08/12)

Contents

Weekly Seminar

  • Summer school: we will organize a barbecue and drinks, therefore some stuff has to be bought.
  • We now have contact to the first sewage treatment plant.
  • Malak will translate our abstract into english for the CAS conference in munich.

Monday July 2nd

  • Team Student Academy:
    • During the whole week the two presentations for school academy were prepared and the barbecue for the pupils was organized. In the lab the last preparations were made.
  • Team Fungal and Plant Laccases:
    • 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.

Tuesday July 3rd

  • Team Site Directed Mutagenesis:
    • Decided how to insert silent mutations to get rid of the restriction-sites with an eye on the codon-usage of host- and scource-oragnism, using the Kazusa “Codon Usage Database”
    • ecol’s illegal NgoMIV will be deleted by changing ggG to ggA (Glycin) at 2307
    • tthl’s illegal PstI will be deleted by changing caG to caA (Glutamine) at 2796
    • bpul’s illegal XbaI will be deleted by changing ctA to ctT (Leucin) at 2883
    • bpul’s mutation will be deleted by changing Gag (Glutamat) to Aag (Lysin) at 2317
    • xccl’s first illegal PstI will be deleted by changing ctG to ctC (Valine) at 2247
    • xccl’s second illegal PstI will be deleted by changing ctG to ctC (Valine) at 3633

Wednesday July 4th

  • Team Student Academy:
    • Final meeting with all instructors of the student academy to talk about all details.
  • Team Cloning of Bacterial Laccases:
    • After the failed attempts to isolate the laccase genes from Streptomyces lavendulae and Streptomyces griseus we assumed that maybe the sequences from this laccases and the laccase sequences we designed the primers for were to different to get a product (look here for more details). So we used the sequences of the laccase genes we tried to isolate from the Streptomycetes strains and blasted them against an intern database from our university. The results showed that we could use genomic DNA from Streptomyces sp. tuebingen, S.roseochromogenes and Streptomyces sp. goettingen, from which we can get already isolated genomic DNA. Therefore we designed primers for the isolation of the predicted laccase genes from these three strains.
  • Team Shuttle Vector:
    • Phusion PCR was done with the following primers to see if the genomic DNA can used as template for the part 3OAX1. For creating the mating factor alpha 1 K863206 the plasmid pPICzA was used, because we did not have the wildtype strain of Saccharomyces cereviceae. For protein secretion we used the mating factor alpha 1 from Sacharomyces sereviceae because it secrete protein in the outer media better than the secretion factor from P. pastoris (LITERATURANGABE)
Used primers Template Successful amplification For BioBrick part
3'AOX_FW and 3'AOX_RV X-33 yes K863201
3'AOX_FW and 3'AOX_RV GS115 yes K863201
Ko_alpha_FW and Ko_alpha_RV pPICzA yes K863206
tAOX1_FW and tAOX1_RV X-33 yes K863203

Thursday July 5th

  • Team Shuttle Vector:
    • Some fragments for Gibson Assembly with overlapping DNA sequences were amplified via Phusion-PCR. The fragment 5'AOX1 was amplified with the primer pair pSB1C3-5aox1-f and pSB1C3-5aox1-r. The fragment MF-alpha1 was amplified with the primer pair 5aox1-mfalpha1-f and 5aox1-mfalpha1-r. The fragment 3'AOX1 was amplified with the primer pair his4-3aox1-f and his4-3aox1-r. The fragment pSB1C3 was amplified with the primer pair 3aox1-pSB1C3-f and 3aox1-pSB1C3-r.
  • Team Shuttle Vector:
    • Phusion PCR was done with the following primers to create fragments with 5' overlap for the Gibson assembly. Also a gel purification was done.
Fragment Used primers Template Successful amplification
5AOX1 pSB1C3-5aox1-f and pSB1C3-5aox1-r X-33 yes
Kozak + MFalpha1 5aox1-mfalpha1-f and 5aox1-mfalpha1-r X-33 yes
3AOX1 his4-3aox1-f and his4-3aox1-r X-33 yes
pSB1C3 3aox1-pSB1C3-f and 3aox1-pSB1C3-r RFP::pSB1C3 yes

Friday July 6th

  • Team Database:
    • FINISHED!! We finished our database. The search statement is now correct. And here it is:
Figure 1: Finally our saerch statement.


Saturday July 7th

  • Team Fungal and Plant Laccases:
    • We received an e-mail from the working group leader Prof. Dr. Bornscheuer (Institute of Biochemistry, Dept. of Biotechnology & Enzyme Catalysis at Ernst-Moritz-Arndt-University in Greifswald (Germany)). He send us a confirmation that the plasmids with the cDNA of four Trametes versicolor-laccases and one laccase of Pycnoporus cinnabarinus were sended to the iGEM Team Bielefeld.

Sunday July 8th

Summary of Week 11

Week 11 (07/09 - 07/15/12)

Contents

Monday July 9th

  • Team Student Academy:
    • This week the Student Academy took place. Today we held a presentation about the background of our experiments and answered all the questions the pupils had. Furthermore two of us participated at the an unconstrained meeting between the instructors and the pupils in the evening.
  • Team Shuttle Vector:
    • Phusion PCR was done with the following primers to create fragments with 5' overlap for Gibson assembly. Also a gel purification was done.
Fragment Used primers Template Successful amplification
his4 promoter taox1-phis4-f and taox1-phis4-r X-33 no
Kozak + his4 phis4-kozak-his4-f and phis4-kozak-his4-r X-33 no
    • Because no PCR fragments could be detected. The same PCR was running with 10 µL of 5M Betain in a sample of 50 µL and provide to the results, that no PCR fragment for the fragment pHIS4 could. But the fragment Kozak + his4 with an 5' overlap could be detected.
  • Team Site Directed Mutagenesis: Council with Katharina Thiedig, who did the Site Directed Mutagenesis for the last years iGEM-Team-Bielefeld, about how to use the “QuikChange Primer Design”-program and the SDM-protocol
  • Team Cellulose Binding Domain:
    • Talked to Dominik Cholewa from the fermentation group of Bielfeld University about cellulose binding domains and got confirmed that they have some on plasmid and want to share them with us.

Tuesday July 10th

  • Team Shuttle Vector:

Surprisingly, why the PCR for the fragment his4 promoter does not raise the theoretical fragment with X33 as template, we check the DNA sequence we used for primer design. For design the primer taox1-phis4-f and taox1-phis4-r we used the DNA sequence from the BioBrick BBa_K563000. But this part was designed on DNA from Sacharomyces cereviceae. So we thought that we can not use this DNA sequence in Pichia pastoris. Our concerns were that the promoter site could not be detected by the transcriptional regulatory machinery. We tried to identified the promoter region upstream the his4 gene on the plasmid pPIC9K from Invitrogen. Some blasts later, we saw that the his4 ORF from this plasmid is virtually the same as the CDS in NCBI. Therefore, to create primers we used the DNA sequence from the plasmid pPIC9K and used the genomic DNA from the wild type P. pastoris X-33 or PCR amplification. Thus the primers taox-his4-f, taox-his4-r and his4-3aox1-f02 were new designed and ordered.

  • Team Site Directed Mutagenesis: Generated primer-sequences with Agilent Technologies “QuikChange Primer Design” for bpul, xccl, ecol and tthl and named the primers by their source-organism and place of mutation flanked on the left side by the original base and on the right by the mutated base.
    • ecol-g2307a
    • tthl-g2796a
    • bpul-a2883t
    • bpul-g2317t
    • xccl-g2247c
    • xccl-g3633c
  • Team Cellulose Binding Domain: Searched NCBI for Cellulose, Chitin & Keratin binding motifs in accessible organisms; found two Chitin-binding-domains in Bacillus halodurans
  • Team Student Academy:
    • The experiments were performed by one half of the pupils in groups of 2-3. Afterwards we held a presentation about the iGEM competition, the project of the last two teams from Bielefeld and about our project. In the evening we had a barbecue with the pupils and the iGEM team with a lot of interesting discussions.
  • Team Fungal and Plant Laccases:
    • We got the sequences for five different fungal laccases from the requested plasmids from Monday 2nd and they wanted to send us plasmids which contain the cDNA sequences of five different laccases from Trametes versicolor and Pycnoporus cinnabarinus.
      • tvel5 from Trametes versicolor
      • tvel10 from Trametes versicolor
      • tvel13 from Trametes versicolor
      • tvel20 from Trametes versicolor
      • pcil35 from Pycnoporus cinnabarinus
    • We designed primer pairs with prefix and suffix overhanging ends for cloning in pSB1C3 and a primer pair for cloning in shuttle vector.
    • The first primer pairs were designed with standard prefix and suffix sequence and 20 bases complementary to the start and end of the ORF sequences.
    • Additional primer pairs 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 designed with the last 20 bases of the laccase genes and a terminator overhanging end and a AarI restriction site.

Wednesday July 11th

  • Team Cellulose Binding Domain: Looked the sequence of Clostridium cellulovorans cellulose binding protein gene (cbp A) up and made a Clonemanager-file. We used a protein-BLAST of the translated protein-sequence to find the location of the cellulose binding domain within the protein: We found it should be from base 103 to base 378 of the open reading frame.
  • Team Student Academy: The experiments were performed by the second half of the pupils and the groups from yesterday analyzed their plates.
  • Team Cloning of Bacterial Laccases: We made glycerin cultures of the finished BioBricks.
  • Team Shuttle Vector: The gel purified fragments 3AOX1 (K863200), MFalpha1 (K863206), pAOX1, tAOX1 (K863203) and the plasmid pSB1C3 were double digested with EcoRI and SpeI as in the follow. Mix 10 µL of PCR amplification and pSB1C3, respectively to 18 µL bidest. water, add2 µL of 10x buffer O, 0,5 µL of each enzyme. After incubation for 1 h at 37 °C and inactivation at 80 °C for 20 min the plasmid was dephosphorylised with Shrimp Alkaline Phosphatase (SAP) by adding 1 µL SAP to the samples. The ligation was done with an equimolar ratio of plasmid to insert of 1:3 and at presence of 2 µL 10x ligase buffer and 1 µL of T4 ligase enzyme. After incubation at room temperature for 1 h, the cells were transformed in E. coli KRX cells. But the transformation was not successful.

Thursday July 12th

  • Team Shuttle vector PCR for more amplification product of 3AOX1 for the part shuttle vector (K863204) and 5AOX1 for the part K863200 was done as described before.

Friday July 13th

  • Team Student Academy:
    • The groups from Wednesday analyzed their plates. Together with the pupils we made a final analysis of the results, discussed about all problems and questions and helped with the preparation of a presentation, they had to hold in front of all instructors and participants.
  • Team Shuttle Vector> Phusion PCR of the fragment tAOX1 that contains upstream an bidirectional double AarI restriction site was done with the wild type X-33 and GS115 as DNA template. The positive control was as ever positive, but no other amplicons.
  • Team Cloning of Bacterial Laccases:
  • We got new Streptomyces DNA from or supervisor C. Rueckert. He did an alignment with our S. griseus and S. lavendulae sequences against a local database for Streptomyces and identified S. rosechromogenes, S. tuebingen and S.goettingen. Those laccase genes showed simularity to our laccase genes. Since he had isolated chromosomal DNA we were able to work with them. We set the PCR with the three Streptomyces. On this PCR S. tuebingen DNA could be amplified, but it wasn't specific for that what we expected. No products could be amplified on the other two.

Saturday July 14th

Sunday July 15th

Summary of Week 12

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

Contents

Weekly Seminar

  • For our sponsoring we sent all the treaties, to all our sponsors so far.
  • Julia V. will organize a guided tour to a sewage treatment plant.
  • The summer school was a great success and all the pupils appreciated our organization and the experiments.
  • Julia V. will be responsible for the wiki design from now on.
  • A big question appeared: How can we put our database on the wiki?
  • We will take new team photos next week.
  • For the safety/security everyone has to read the safety regulations regarding the organisms and chemicals.
  • Next week there will be a short presentation about all used organisms and their laccases
  • Agatha and Saskia will create templates for our webpages.
  • Nadine will gather information about places to stay in Amsterdam and how to get there.
  • Everyone has a passport?
  • Gabi will present the judging criteria next week.
  • Julia is looking for a BioBrick, which we could use and improve.
  • Isabel created a poster for the conference in Munich, everyone has to review it.

Monday July 16th

  • Team Fungal and Plant Laccases:
    • After we forgot to delete the signal peptide sequences, which are present in the fungal laccases we designed new forward primers for the laccases tvel5, tvel10, tvel13, tvel20 and pcil35 with the overhanging ends for cloning in our shuttle vector. Trametes laccases have a signal sequence after the start codon. This signal peptide we now delete by taking the first 20 bases after this sequence in our FW primers.
    • 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 diluted cDNA was too low. We will do some washing and try again.
  • Team Cellulose Binding Domain:
  • Team Cultivation & Purification:
    • We searched for some information for the best cultivation conditions in the internet. We found an interesting report of the Deutsche Bundesstiftung Umwelt (DBU) containing some interesting facts about different laccases as for example that the bacterial laccases are toxic to the bacterias, so that the production could be better under oxygen limitation and reduced temperature. Based on this article we decided to test flask with and without baffles and different temperatures.
    • We prepared the basic media and solutions we will need in the lab.
    • Note: All following BioBricks are cloned into pSB1C3 and therefore cultivated with 20 µg/mL chloramphenicol unless otherwise specified! Cultivations of E. coli KRX without plasmid will be performed without antibiotics.

Tuesday July 17th

  • Team Site Directed Mutagenesis: Made Clone Manager files for the Trametes versicolor laccase plasmids we got send and analyzed them:
    • tvel5:
      • one silent mutation (at 154 bp) and one amino acid alternating mutation at 227 bp (G for A, replacing D with N), a third was claimed to be at 1559 but sequencing showed it wasn’t.
      • No illegal restriction-site for the Silver-assembly, but three Freiburg-assembly restriction-sites: two NgoMIV- and one AgeI-restriction-sites
    • tvel10:
      • eight silent mutations 171, 444, 1020, 1173, 1239, 1443, 1485 & 1503 bp
      • Two amino acid alternating mutations (1048 bp C for T, replacing F with L and 1078 bp A for G, replacing D with N)
      • Two PstI-restriction-sites (One in the signaling-sequence at 7 bp and one at 1160) and one SpeI-restriction-site at 241 bp
      • One Freiburg-assembly restriction-site (AgeI at 912 bp)
    • tvel13:
      • 56 silent mutations
      • Three amino acid alternating mutations and one whole codon is missing at the very end
      • One EcoRI- and one PstI-restriction-site
      • Two Freiburg-assembly restriction-sites (two NgoMIV-sites)
    • tvel20:
      • about 32 silent mutations, three amino acid alternating mutations and four Freiburg-assembly restriction-sites (one AgeI, three NgoMIV)
    • pcil35:
      • no illegal restriction-site
  • Team Cultivation & Purification:
    • We prepared our first precultures of E. coli KRX as negative control and E. coli KRX containing BBa_K863005, BBa_K863020, BBa_K863015 and pBpL6.
    • Note: We will cultivate E. coli KRX with pBpL6 until we will get the laccase ORF with T7 promotor and His tag in the same pSB1C3 vector as the other BioBricks. To cultivations of E. coli KRX with pBpL6 we always will add 100 µg/mL ampicillin.

Wednesday July 18th

  • Team Site Directed Mutagenesis: Made Primer-Mixes for the bacterial laccases. Set Pre-culture of XL1 blue. Got everything ready for Lab work.
  • Team Cloning of Bacterial Laccases:
  • We diluted our chromosomal DNA to a concentration of 20 ng/µL, since the volume we got was to low for doing many PCRs and did again a PCR reaction. This time S. goettingen laccase DNA could be identified but not S. tuebingen. The reaction conditions were the same so we were surprised because S. tuebingen didn`t work.
  • Team Fungal and Plant Laccases: Some of the ordered parts from the Parts Registry arrived and we plated the biobricks BBa_K500000, BBa_K500001, BBa_K500002, BBa_K500003 and BBa_K392014. Above all we are interested in BBa_K500002 because it’s a codon optimized laccase from Trametes versicolor and we want to use this laccase in our P. pastoris shuttle vector and characterize it.
  • Team Cultivation & Purification:
    • Today we performed our first flask cultivation. We cultivated E. coli KRX with BBa_K500005, BBa_K863020, pBpL6 and BBa_K863015 and as negative control we used E. coli KRX.
      • Settings: We used 300 mL flasks without baffles, final volume: 60 mL, autoinduction medium, 30/37 °C, durance: 24 hours
    • Found out that we had a mixed culture of E. coli KRX with pBpL6, because it grew on chloramphenicol, but has only an ampicilline resistance. So we could not use this culture.

Thursday July 19th

  • Team Site Directed Mutagenesis:
    • Made electrocompetent XL1 blue cells. Made pfu-PCR of the bpul-plasmid with bpul-a2883t primer-mix, the ecol-plasmid with ecol-g2307a primer-mix, the xccl-plasmid with the xccl-g2247c primer-mix and tthl-plasmid with the tthl-g2796a primer-mix. Digested the template with DpnI.
    • Generated Primers for silent mutations of tvel10 illegal restriction-sites:
      • the illegal SpeI will be deleted by changing acT to ggA (Threonine) at 243 bp of the know sequence
      • the illegal PstI will be deleted by changing ccT to ccA (Proline) at 1161 bp of the known sequence
      • one illegal restriction site in the signaling-sequence can not be mutated, since it is to close to the beginning of the known sequence (7 bp) and also it is not essencial, because the gene will be used without the signaling sequence
  • Team Cellulose Binding Domain:
    • Gathered some information about carbohydrate binding domain X2 (which is a more common domain in the organisms we handle than a cellulose Binding Domain).
    • Used NCBI Nucleotid BLAST on Status: 500

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(the Cellulose-binding motif from C. josui Xyn10A gene) and it 100% fits to 1526 bp to 2608bp of AB041993.1 (Clostridium josui xynA gene for xylanase A).
    • Used NCBI Protein-BLAST on AB041993.1 and found one cellulose binding domain and one carbohydrate binding domain within the protein.:
      • pfam02018: CBM_4_9 (Carbohydrate binding domain) from AS 193 to AS 332 - corresponds to 1088 bp - 1507 bp in the gene and is not in the coding sequence of Status: 500

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.

      • cd09619: CBM9_like_4 (DOMON-like type 9 carbohydrate binding module) from AS 716 to AS 887 - corresponds to 2657 bp to 3172 bp in the gene and is also not in coding sequence of Status: 500

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.

      • Given sequence of Status: 500

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(1589bp to 2590bp) does corresponds to the sequence of the Glycosyl hydrolase 1589 bp  to 2590 bp and is not one of the binding motifs. The whole review can be read at the Experience-page of BBa_K392014.
  • Team Cloning of Bacterial Laccases:
  • S. goettingen and S. tuebingen DNA were cleaned up and we done an enzymatic digestion to ligate it into the pSB1C3 vector. We did the digestion with EcoRI and SpeI.
  • Team Fungal and Plant Laccases: Colonies from plated BioBricks from 18.07 were spread on nutrient agar for plasmid isolation.
  • Team Cultivation & Purification:
    • The cultures of 07/18 were centrifugated, cells were disrupted via sonication in the special buffer for each laccase and after another centrifugation the supernatant was given to the activity test team.
    • Made precultures analogous to them on 07/17.

Friday July 20th

  • 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. Bülter form the clarification plant Schloß Holte (near Bielefeld) and he invited us to present our project.
  • Team Shuttle vector: Gel purification of the fragments Kozak-MFalpha1 K863206 and 3'AOX1 K863201 was done.
  • Team Site Directed Mutagenesis:
    • Transformation of XL1 Blue with the PCR products (see day before)
  • Team Cellulose Binding Domain:
    • Used Protein-BLAST on the translated sequence of the Clostridium cellulovorans cellulose binding protein, the Bacillus halodurans strain Cochin chitinase GU481106.1 and chitin-binding protein [Bacillus halodurans C-125] BAB05022.1, to get more information about the predicted sequences of Carbohydrate binding domains.
    • Looked up possible linkers in the Parts Registry:
      • 2 aa GS linker: BBa_J18920 [GS]
      • 10 aa [GS]x linker: Status: 500

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[GSGSGSGSGS]
      • 15 aa flexible glycine-serine protein domain linker; Freiburg standard 1 Star: Status: 500

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[GGGGSGGGGSGGGGS]
      • 10 aa flexible protein domain linker 1 Star: Status: 500

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[GENLYFQSGG]
    • Made a few possible primers for CBDclos with T7 and RBS (Status: 500

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), a Freiburg-Suffix and possible linkers

    • Wrote an E-Mail to Jun.Prof Thorsten Seidel (FRET-Expert of Bielefeld University) asking him about the linkers they use for fusing GFP for FRET.
  • Team Fungal and Plant Laccases: Isolating the plasmids BBa_K500000, BBa_K500001, BBa_K500002, BBa_K500003 and BBa_K392014. To be sure that in every plasmid contains the correct part we made a control restriction with NotI. It showed that all parts are on the correct hight in agarose gel.
  • Team Cultivation & Purification:
    • Another flask cultivation was made analogous to the one on 07/18 but at 23 °C.

Saturday July 21st

  • Team Cellulose Binding Domain:
    • Made primers for GFP_His (using BBa_I13522 as template)adding a C-termial His6tag for easy extraction
    • Jun.Prof Thorsten Seidel answered to the mail of yesterday and said that GFPs are easy to fuse and that they use linkers with four AS:
      • ccg gtc gcc acc upstream of the GFP
      • gaa agc ggc cgc downstream of the GFP; Which is will work fine, even with a proline in it's sequence.
    • We decided to use four linking AS also and chose to add a C-terminal Glycine-Serine-Linker (BBa_J18920) to the cellulose binding domain, which would be four linking AS with the Freiburg-scar (Threonine-Glycine).
  • Team Cloning of Bacterial Laccases:
  • Since our PCRs didn't work and the Streptomyces laccases have more then 70% GC content we changed the dNTP concentration to 10 mM per base. Our gelelectrophoresis showed us that again S. goettingen has a product in correct length so we did again a PCR with same conditions. The rest of this product was cutted from an agarose gel and set ligation with the pSB1C3 vector and transformation. Because the isolated DNA concentration was to low the transformation showed no positive results.
  • Team Cultivation & Purification:
    • Culture from 07/20 was harvested and the cells were disrupted via sonication.

Sunday July 22nd

  • Team Shuttle Vector:
    • To get more amplicon from the part 5AOX1 K863200 an other Phusion PCR was done and the PCR product gel purified.
    • To get the tAOX1 fragment for the shuttle vector, we run a PCR with the KOD DNA polymerase as descriped in the protocol.
    • While debugging and finding an solution for PCR fragment for the part tAOX1, test the annealing temperature of 60 °C that was proposed by the Tm calculator from NEB. Therefore, we run a temperature gradient from 50 °C until 67 °C with the usual protocol for Phusion DNA polymerase. But no specific product could be detected.
    • Because there were also many problems to get a PCR fragment of the HIS4 fragment for the shuttle vector we run a temperature gradient from 55 °C until 60 °C with the usual Phusion DNA polymerase protocol. From 50 °C until 65 °C we could detect the right fragment. The highest yield was at 60 °C.
  • Team Cellulose Binding Domain:
    • We decided to use the exact sequence of BBa_J18920 (GGCAGC) of the linker in the partsregistry.
    • Checked the CBDs we had access to for illegal restriction sites and found none, not even NgoMIV or AgeI. So we decided to use a Freiburg-Assembly to build the CBD-GFP fusion protein.

Summary of Week 13

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

Contents

  • From 07/23 - 07/25/12, some of our team members participated at the CAS conference in Munich.

Weekly Seminar

  • we will attend at the Biolympics it Münster, hosted by the bts just to have some fun.
  • We decided to write down every result in the labjournal, so that problems and their solution can be understood and reconstructed

Monday July 23rd

Activity of ECOL, BHAL, BPUL and XCCL, measured in the supernatant of cells cultivated at 30°C via oxidized ABTS depending on time. E.coli KRX without plasmid functiones as a negative control. Values are calculated by taking the average out of 4 measurements (n=4).
Activity of ECOL, BHAL, BPUL and XCCL, measured in the supernatant of cells cultivated at 37°C via oxidized ABTS depending on time. E.coli KRX without plasmid functiones as a negative control. Values are calculated by taking the average out of 4 measurements (n=4).
  • 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 E.coli KRX that contained the plasmid to produce ECOL, BPUL, BHAL, XCCL or E. coli KRX (negative control) and measured as usual. There was no change in the OD so that we assume that no secretion takes place.
  • Team Shuttle vector:
    • Electroporation of RFP::pSB1C3 was done to get more plasmid DNA, but the wrong competent cells and selective plates were choosen.
    • Phusion PCR of the tAOX1 part for the shuttle vector was done without betaine addition with the genomic DNA of P. pastoris GS115 and the wild type X-33. But only a small unspecific product was visible.
  • Team Cloning of Bacterial Laccases: The transformation from the 21st July showed no results. The plates were unfortunately empty. So we did an another PCR. This time the Annealing Temp. was set to 56°C.
  • Team Fungal and Plant Laccases: Successful PCRs of laccase gene Tv5 from Trametes versicolor with plasmid DNA from Uni Greifswald as template.
  • Team Site Directed Mutagenesis: Transfered 4 colonies of each transformation to an new dish for plasmid-isolation.



Tuesday July 24th

  • Team Shuttle Vector: To get the fragment tAOX1 for the shuttle vector the Phusion PCR conditions were changed. A two-step PCR was done with the following conditions: After initial denaturation at 98 °C for 6 min, the first 10 cycles were run at: denaturation at 98 °C for 15 s, primer annealing at 55 °C for 30 s, elongation at 72 °C for 30 s. The following cycles were run at: denaturation at 98 °C for 15 s, primer annealing at 67 °C for 30 s, elongation at 72 °C for 30 s. The final elongation run at 72 °C for 5 min. But the PCR ghost was not satisfied and the right amplicons could not be detected. Just some unspecific small fragments could be detected in the geleletrophoresis.
  • Team Site Directed Mutagenesis: Plasmid-isolation of all 16 different colony-dishes
  • Team Fungal and Plant Laccase: Since our cDNA is now ready to go and also our primers already arrived we started a PCR. Check protocols for more information about the exact setup. Unfortunately no bands could be seen via gel electrophoresis. We blamed the high salt concentration and decided that a some cleaning via ethanol precipitation might be a solution to our problem.
  • Team Activity Tests: After testing the supernatant of the cultivations yesterday we got to the more exciting part today. We tested the cell extract for laccase activity by filling each well with 140 µL cell extract, 40 µL buffer, 18 µL H2O and added 2 µL ABTS. Unfortunately no activity was seen. We felt like we could not take the laziness of our laccases. We thought and discussed what the reason could be for them to be inactive.
Fig. 1: Check for active BPUL laccases in the cell extract of the last cultivation at 23°C, 30°C and 37°C As well compared
Fig. 2: Check for active ECOL laccases in the cell extract of the last cultivation at 23°C, 30°C and 37°C (n=4).
Fig. 3: Check for active BHAL laccases in the cell extract of the last cultivation at 23°C, 30°C and 37°C (n=4).
Fig. 4: Check for active XCCL laccases in the cell extract of the last cultivation at 23°C, 30°C and 37°C (n=4).
Fig. 5: Cell extract of the last cultivation of E. coli KRX without plasmid at 23°C, 30°C and 37°C as a negative control (n=4).




    • Is there something wrong with the transformed construct, maybe a mutation?
    • Was the laccase synthesized but is inactive for some reason?
    • Was the laccase produced but not in a high enough amount so that we can´t detect its activity in the first place?

Another thought was that there might be copper missing. Since laccase is a copper-enzyme it might be necessary to add copper to the medium whenever it is produced in such high amounts. It´s late, so we will do that tomorrow.


Wednesday July 25th

  • Team Site Directed Mutagenesis: Test-digestion (with the enzym of the mutated restriction-site) of the Plasmids from the 16 different colonies revealed that two bpul, one ecol and two tthl mutants have the right bands in the gel and seem be mutated correctly. Xccl-colonies are all unmutated (no Band at 3636).
    • pfu-PCRs with the two correct bpul-plasmids as template and the bpul-g2317t primer-mix
    • two new pfu-PCRs with the xccl-plasmid one with the g2247c primer-mix and the other with the xccl-g3633c primer-mix
    • Prepared all positive colones for sequencing
  • Team Cultivation & Purification:
Cell extracts from cultivations were incubated with 2 mM copper sulfate for 2 h and measured for activity via OD420.
Cell extracts from cultivations were incubated with 4 mM copper sulfate for 2 h and measured for activity via OD420.
  • Team Activity Test: Today is copper day! First we divided each sample (the ones from yesterday) and added 2mM or 4mM copper sulfate to the halfs, respectively. We incubated our samples for 2h and hoped that the concentration gradient would cause the laccases to exchange the ion they have integrated instead against copper. We measured their activity again after those 2h but there was no change seen. Team Cultivation plans to add copper from the very beginning of cultivation next time.





Thursday July 26th

  • Team Modeling and Team Sponsoring: meeting Mr. Bülter from the clarification plant of Schloß Holte and finding a new cooperation partner. He wants 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 project.
  • Team Site Directed Mutagenesis: Went on with the PCR products of xccl and bpul, transformed them into XL1 Blue and plated them on select-agar
    • pfu-PCRs with the tvel10-plasmid, one with the tvel10-243 primer-mix and one with the tvel10-1161 primer-mix. Both PCRs showed correct bands for the PCR-product in a test-agarose-gel-electrophoresis. Digested template with DpnI.
  • Team Fungal and Plant Laccases: We did PCR on Trametes versicolor laccase tvel5 and Pycnoporus cinnabarinus pcil35 with Tv_lac10.P.FW / Tv_lac10.S.RV and Pc_lac35.P.FW / Pc_lac35.S.RV primers. Additionally we want the laccases with overhanging ends for cloning in our shuttle vector for expression in Pichia pastoris. Therefore we used Pc_lac35_FW_oS / Pc_lac35_RV and Tv_lac5_FW_oS / Tv_lac5_RV primer pairs.
  • Team Cultivation & Purification:
    • Glycerine cultures were made of all given BioBricks, to use them for precultures.
    • Made new precultures analogous to 07/25, as well as a preculture of E. coli KRX with BBa_K863010.
    • Found out, that it could be important to add CuCl2 to the medium, because the copper is important for the active center of the laccases and to improve their stability.

Friday July 27th

  • Team Site Directed Mutagenesis: plated four colonies of each transformation-dish (xccl & bpul) on a petri dish for plasmid-isolation. Purified the digested PCR-products of tvel10.
  • Team Cellulose Binding Domain:
    • We made the decision to only use cellolose binding domains (CBDs) and not carbohydrate binding domains of any kind to keep them comparable. This means we won't use the binding domains of Bacillus halodurans.
    • Redesigned Primers for CBDclos - added the bases TA in between RBS and ATG since the RBS can be made stronger by adding more adenines in the sequence upstream of the RBS.
    • Made similar primers for CBDcex (the CBD of the Cellulomonas fimi exoglucanase we got for the fermentation-group of Bielefeld University)
    • Checked the Wiki of the iGEM 2012 Osaka University-Team about their Status: 500

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BioBrick which should be a binding motif, but is the glycosyl hydrolase domain, for their intentions. We guess they really messed it up. It would have been nice to have a CBD from the partsregistry.
  • Team Fungal and Plant Laccases: The PCR on tvel5 laccase was positive after the first trial for both primer pairs. For the tvel35 laccase the PCR didn’t work.
  • Team Cultivation & Purification:

Saturday July 28th

Activity Test of different Laccases cultivated with 0.25 mM CuCl. Cultivation has taken place using different methods. 'os' stands for a cultivation without chicanery, 'ms' for a cultivation with chicanery. Numbers (60, 200) indicate the volume of cultivation. No oxidation of ABTS trough our produced laccases could be measured.
  • Team Shuttle Vector:
    • Because the first Gibson assembly failed, more fragments with a higher concentration are necessary. All parts were amplificate by the same protocols as described.
    • Also the Part HIS4 K863202 was amplified as described.
    • And the Part tAOX1 K863203 was amplified by the same conditions as the fragment tAOX1 for the shuttle vector.
  • Team Activity Tests: See, we are back already! Today we received different samples from ECOL, BPUL, BHAL, TTHL, XCCL 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.
  • Team Fungal and Plant Laccases:
    • Purification of Trametes versicolor tvel5 PCR product.
    • With the cDNA samples we got out of the siliques of A. thaliana we did two PCRs. After waiting a hour for the PCR to stop, we started a gel, hoping for really thick bands. But nonetheless there were non. Also the PCR with actin primers didn't give a result. A possible reason for this could have been contaminations like salts in our samples. Before translating the mRNA into cDNA we measured the following properties: 260/280: 1.93 and 260/230: 1.28. This indicates a high salt concentration which could be interfering during the PCR. For the next PCR we want to wash our cDNA and hope for better results with a clean and pretty sample.
  • Team Cultivation & Purification:
    • Cells of cultivation 07/27 were disrupted via sonication and given to the activity test team.

Sunday July 29th


Summary of Week 14

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

Contents

Weekly Seminar

  • From now on, there will be two weekly meetings: monday, 12.15 and thursday 9.00.
  • Nadine sent all of us a poll, answers will be used on the wiki.
  • For picture uploads we will use the name: Bielefeld2012_x.
  • Which sewage treatment plant uses activated carbon in the third stage of treatment?
  • On 25th of august, all german teams will host the SynBioDay. In Bielefeld we will cooperate with Bielefeld Marketing and to raise public interest we will call it StreetScience. As part of our collaboration we will invite the iGEM team from SDU-Denmark to help us with the organization and the realization of this common day.
  • On StreetScience we will have a little experiment about extracting fruit DNA with an easy method.
  • Do we want to travel to Amsterdam by bus or train?
  • Do we want a ship as our accommodation in Amsterdam?
  • Gabi has presented the judging criteria including the bronze, silver and gold criteria as well as the special prices.

Monday July 30th

  • Cloning of Bacterial Laccases:
    • The PCR products from the 23th July was digested with EcoRI and SpeI same as the pSB1C3 vector. Then the ligation was set and transformed into competent E. coli KRX cells.
    • Since we haven't got any more PCR products we set a PCR. Only S. goettingen worked.
  • Team Fungal and Plant Laccases: Digestion of tvel5 PCR product with prefix and suffix ends for cloning in pSB1C3 and digest of laccase with the overhangs for cloning in shuttle vector. Additionally we did the PCR on tvel35 with both primer pairs again. This time we lowered the annealing temperatures and got products with both primer pairs.
  • Team Cellulose Binding Domain:
    • Redesigned the primer-sequences another time giving the CBDs a few additional amino acids from the by Protein-BLAST predicted domain. CBDcex: 4 AS N-terminal, 2 C-terminal. CBDclos: 2 N-terminal (starting with a natural ATG) and 2 AS C-terminal.
    • Prepared Status: 500

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for Sequencing
  • Team Site Directed Mutagenesis: Plasmid-isolations of all the bpul- and xccl-mutants.
  • Team Cultivation & Purification:
    Figure 1: Growth kinetics of cultivation of E. coli KRX with BBa_K863005, BBa_K863020, BBa_K863010, BBa_K863015 and pBpL6. Parameters: autoinduction medium, 0.25 mM CuCl2, 28 °C.
    • We made the SDS-Pages for the cultivation from 07/27, but they did not seem to be promising.
    • We started another cultivation of E.coli KRX with BBa_K863005, BBa_K863020, BBa_K863010, BBa_K863015 and pBpL6.
      • Settings: 300 mL flasks without baffles, final volume: 60 mL, autoinduction medium, 0.25 mM CuCl2, 28 °C.
      • A growth kinetics was recorded every 45 minutes.
    • We decided that we also need a positive control for the next cultivations, to see if our autoinduction medium works. We chose BBa_K525710. Furthermore we will make cultivations in which we manually induce the expression after 4 hours.
    • Made a preculture of E. coli KRX with BBa_K863005, BBa_K863020, BBa_K863010, BBa_K863015 and pBpL6 as well as with BBa_K525710. We used E. coli KRX as negative control.


Tuesday July 31st

  • Team Shuttle Vector
    • To get the tAOX1 fragment for the shuttle vector, we run a primer gradient from 0.1 µM to 1 µM in 0.1 µM steps with a primer annealing time of 15 s and 20 s and the usual Phusion PCR protocol. No fragments could be detected.
    • A DNA template gradient from 10 ng to 250 ng in 50 ng steps was choosen. But no fragment could be detected. Also the primer annealing temperature was varied in 15 s and 20 s. But only small unspecific fragments with from 100 bp to 150 bp could be detected.
  • Team Cloning of Bacterial Laccases:
    • After our laccases are not produced or not detectable we decided to try to express the laccases with a constitutive promoter. Therefore we searched for appropropriate promoters and found different promoters from the Anderson promoter family. We picked three different promoters with different promoter strengths. We chose the parts BBa_J23103, BBa_J23110 and BBa_J23117 and for all three promoters the RBS BBa_B0034. The primers were designed that the FW and the RV primers anneal together to a short oligonucleotide with overhanging restriction site ends for EcoRI and SpeI. So we don’t have to cut the annealed primers, because the sites should appear with correct annealing of FW and RV primers. The goal is to clone the different promoters, the PCR products with the different laccase genes with His-Tag in pSB1C3 in one ligation step. Furthermore we want to exclude the possibility that the His-tag is the reason for no activity, so we also want to clone the gene sequences without a His-tag under control of a constitutive promoter in pSB1C3.
    • Additionally we want to produce the constructs with a new T7 promoter. After our laccases were not expressed we now think that maybe the RBS Status: 500

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, which we changed in our primers from originally 5' aAagaggagaaa 3' to 5' aGagaggagaaa 3' is not or poorly recognized from the ribosomes in the cells.

    • We have a primer pair from last year with the T7 promoter sequence with the RBS BBa_B0034. The primers can be annealed to an oligonucleotide. After boiling the primers and cooling down there should be an oligonucleotide with an EcoRI and a PstI restriction site. We now want to assemble the promoter the laccase genes and the pSB1C3 vector in one step. The pSB1C3 backbone was digested with EcoRI and PstI, the laccases with XbaI and PstI, and the promoter has the restriction sites SpeI and EcoRI (we have to digest the promoter with SpeI, because the original restriction site is PstI).
  • Team Fungal and Plant Laccases:
    • Purification of tvel35 PCR product and digestion for cloning in pSB1C3 backbone.
    • Ligation of tvel5 laccase with pSB1C3 backbone.
  • Team Site Directed Mutagenesis: Plated four colonies per dish of tvel-t243g and tvel-t1161a for plasmid-isolation.
    • Test-restriction of the xccl-mutants showed that no colony was mutated correctly
    • The second site directed mutagenesis of bpul-g2317t could not be rated by test-digestion, since the mutation at 2317 is not because of a illegal restriction site, but a amino acid alternating mutation.
    • Made four bpul-colonies (with both mutations) ready for sequencing
    • plated a four more colonies of xccl (both SDM-sites) for plasmid-isolation
a) E. coli KRX with BBa_K863005, BBa_K525710, BBa_K863020, BBa_K863015 and pBpL6 were cultivated in LB media and manually induced after 4 hours with 0.1 % rhamnose.
b) E. coli KRX without plasmid was cultivated with LB medium and induced after 4 hours with 0.1 % rhamnose.
c) E. coli KRX with BBa_K525710 was cultivated equally to b)
d) E. coli KRX without plasmid was cultivated with autoinduction medium and 20 µg/mL chloramphenicol as well as 100 µg/mL ampicillin as a control.


Wednesday August 1st

  • Team Site Directed Mutagenesis: Plasmid-isolation of tvel- and the xccl-colony-dishes.
    • Test-restriction with showed correct bands for two tvel and one xccl-colony.
    • Made the correct xccl-plasmid ready for sequencing.
  • Team Cloning of Bacterial Laccases:
    • With the new T7 promoter we started new assemblies. We first want to try the new promoter with ecol. So wedigested our laccase PCR products from ecol with and without HIS tag for suffix insertion with XbaI and PstI, boiled and cooled down the joined T7 primer pairs and digested pSB1C3 backbone with EcoRI and PstI. If the plan works, the parts anneal to our final plasmid. We did the ligation and transformation of the approach into competent E. coli KRX cells.
  • Team Cultivation & Purification:
    • Harvesting and cell disruption via sonication of yesterday's cultivation.

Thursday August 2nd

  • Team Wiki: This morning we met for taking a new picture of our group and individual pictures of everyone. Check out our beautiful team members here.
  • Team Cloning of Bacterial Laccases:
  • There were just red colonies on the plates from the transformation the day before. So we didn't do colony PCR and have to do the assembly again.
  • Our Laccases genes from S. goettingen, S. tuebingen and S. roseochromogenes are around 2kb. The NEB-Phusion has a range of 1kb/30sec so we thought if we set the elongation time higher we might have products for S. tuebingen,S. roseochromogene so we did PCR. But the results showed that it doesn't depend on the elongation time from the Phusion because we haven't had any products.
  • Team Fungal and Plant Laccases: Ligation of tvel5 and pcil35 in pSB1C3.
  • Team Site Directed Mutagenesis: pfu-PCRs; two with the positive tvel-t243g-plasmids as templates and the tvel-t1161a primer-mix and another pfu-PCR with the positve xccl-g2247c-plasmid and the xccl-g3633c primer-mix
  • Team Cultivation & Purification:
    • Made SDS-Pages of cultivation from 07/31.
Figure 4: SDS-PAGES of the lysate, the supernatant, and with urea treated pellet (cultivation 07/31) of E.coli KRX with BBa_K863005, BBa_K863020, BBa_K863010, BBa_K863015 and pBpL6 as well as with BBa_K525710 (positive control) and E.coli KRX (negative control). 60 mL in 300 mL flasks without baffles, 30 °C. mI = manual induction, -I = without induction, auto-I = auto-induction.

Friday August 3rd

  • Team Cloning of Bacterial Laccases:
  • Digest of ecol PCR products with and without HIS-Tag and ligation with pT7 in pSB1C3. Transformation of this ligations and transformation of BBa_K525710 for Team Cultivation.
  • We did an annealing temp. gradient PCR with the Streptomyces bacteria. But expect 'S.goettingen neighter S. tuebingen not S. rosechromogenes did not work.
Figure 5: Oxidizing activity of Laccases produced on 07/30 measured with ABTS. 'I(-)' and I(+) indicate no or Inducion which took place, respectively. A increase in OD420 couldn't be seen. (n=4)
  • Team Fungal and Plant Laccases: Transformation of tvel5 and tvel35 in pSB1C3 backbone.
  • Team Site Directed Mutagenesis:
    • DpnI-digestion of the pfu-PCR-products (from yesterday). Transformed tevl- and xccl-double-mutants into XL1 Blue and plated them on selection-agar.
    • Sequencing results:
      • Both tthl-g2796a-mutations were successful
      • ecol-g2307a has additional mutations
  • Team Cellulose Binding Domain: Transformed the plasmids p714 (with the CDBcex domain) and p570 (with the CBDclos domain) we got from the fermentation group of Bielefeld University in KRX an plated them on kanamycin-selection-agar.
  • Team Activity Tests: Our lovely Team Cultivation started new cultivations on 07/30 and we got the products. More precisely we got the supernatant of the lysis Team Cultivation did before. We tested 198 µL of each sample with 2 µL ABTS for the activity reaction, but sadly we couldn't detect anything (see Fig. 5).

Saturday August 4th

  • Team Cloning of Bacterial Laccases:
  • We did the transformation of the ligations from August 1st again.
  • Team Site Directed Mutagenesis: Plated four double-mutant-colonies from each transformation (both tvel-mutants and the xccl-mutant on selection-agar for plasmid isolation.
  • Team Cellulose Binding Domain:
    • The transformation of p570 brought up only a few colonies, so we took a few and plated them again on a selection-agar-dish.
    • Plasmid-isolation of p714
  • Team Cultivation & Purification:
Figure 6: Check for active laccases in supernatants from lysed samples of the last cultivation (ECOL,BPUL,BHAL,TTHL and XCCL) (n=4).
  • Team Activity Tests: Another cultivation ended and we are happy to measure the supernatants from the 31st September cultivated samples. We used 158 µL of the supernatant with 40 µL and added 2 µL of 20 mM ABTS to measure some activity (Fig. 6). Nevertheless we couldn't see any increase in absorbency at 420 nm after 5 minutes, indicating, that we have no active laccase in the supernatants.


Sunday August 5th

  • Team Cloning of Bacterial Laccases:
    • The assemblies don't work by now in one step by now, we always get just red colonies from the original RFP plasmid. So we started to clone one part after another in pSb1C3 backbone. For this reason we want to ligate the ecol gene without any promoter in pSB1C3 backbone and in the next step do a prefix insertion with the promoter fragment. Therefore we did the restriction of ecol and ecol_HIS PCR products, the ligation in pSB1C3 and the transformation.
  • Team Site Directed Mutagenesis: Plasmid-isolation of the xccl- and tvel-double-mutants.
    Growth kinetics of E. coli KRX with Status: 500

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Growth kinetics of E. coli KRX with Status: 500

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a) 2 flasks of each culture were inducted with 0.1 % rhamnose after 4 hours of cultivation
b) 2 flasks of each culture were not induced.
  • We recorded the growth kinetics once per hour.


Summary of Week 15

Week 15 (08/06 - 08/12/12)

Contents

Weekly Seminar

Monday August 6th

  • Picking positive colonies from transformation of ecol and ecol_HIS in pSB1C3 for plasmid isolation.

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on selection-agar.

Tuesday August 7th

  • Plasmid isolation and control restriction of ecol and ecol_HIS in pSB1C3 showed correct fragment sizes in agarose gel. So we did a digest for prefix insertion of the new T7 promoter.

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as template and GFP_Freiburg and GFP_His6_compl-primers and clean-up through agarose-gel.

Wednesday August 8th

Thursday August 9th

Friday August 10th

  • Again we did the digest of our new T7 promoter part and the ligation in pSB1C3 backbone with ecol PCR products with and without HIS tag. After that we transformed the ligations in pSB1C3. Additionally we did the same with promoter J23110 instead of T7 promoter.
  • We did PCR on BBa_K863020 with the primers B.halo_FW and B.halo_RV for cloning the gene in pSB1C3 backbone without promoter and HIS tag.
  • We ligated the digested pSB1C3 plasmids with ecol and ecol_HIS with the new pT7 promoter and pSB1C3 backbone and transformed the approaches in KRX.

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for different concentrations of chloramphenicol (20 to 170 µg mL -1. OD600 was measured.

Saturday August 11th

Figure 1: Activity measurement of the supernatant of theBPUL cultivation. Cells were cultivated in different chloramphenicol concentrations. Measurements were done via oxidized ABTS and at OD420.
Figure 2: Activity measurement of the cell extract of theBPUL cultivation. Cells were cultivated in different chloramphenicol concentrations. Measurements were done via oxidized ABTS and at OD420.

Sunday August 12th

Figure 2: Comparison of different lysis methods.Potential laccase activity of ECOL was measured after lysis via OD420.


Summary of Week 16

Week 16 (08/13 - 08/19/12)

Contents

Weekly Seminar

Monday August 13th

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with the GFP_His-primers with positive result.

Tuesday August 14th

Figure 1: TVEL0 activity measured using 0.1 mM ABTS at 25°C over a time period of 5 minutes in presence of different MeOH concentrations (n=4)
Figure 2: TVEL0 activity measured using 0.1 mM ABTS at 25°C over a time period of 5 minutes in presence of different acetonitrile concentrations (n=4)


Wednesday August 15th

Next will be a standard for charts.

Output of enzyme activity measurements via oxidized ABTS and OD420. Measured is the flow through of ECOL, XCCL, TTHL, BHAL and BPUL. E. coli KRX (no plasmid) was used as negativ control and cells containing a plasmid with ligase was used for the control of induction. The elution buffer was as well tested as a negative control.
Output of enzyme activity measurements via oxidized ABTS and OD420. Measured is the activity of ECOL, XCCL, TTHL, BHAL and BPUL. E. coli KRX (no plasmid) was used as negativ control and cells containing a plasmid with ligase was used for the control of induction. The binding buffer was as well tested as a negative control.

Thursday August 16th

Step Condition
Initial denaturation 98 °C, 30 s
Denaturation 98 °C, 10 s
Primer annealing 67 °C, 20 s
Elongation 72 °C, 90 s
Final Elongation 72 °C, 5 min

Phusion PCR was also done for the HIS4 fragment at following conditions with 35 cycles and also PCR product as template.

Step Condition
Initial denaturation 98 °C, 30 s
Denaturation 98 °C, 10 s
Primer annealing 60 °C, 20 s
Elongation 72 °C, 90 s
Final Elongation 72 °C, 5 min

Phusion PCR was also done for the tAOX1 fragment at following conditions with 35 cycles and also PCR product as template. For us it can not be recognized at which annealing temperature the primer binds best. That is why we choose an annealing temperature gradient more. This time from 50 °C until 60 °C. The mixture of the sample is the same as last time.

Friday August 17th

Saturday August 18th

  • Today we performed a ligation from the digestions we have done before. The ligation included of course the T7 promotor, the backbone pSB1C3 and the individual laccase genes. Ligation was performed with T4 ligase for 20 minutes at room temperature and stopped through a five minute incubation at 70 °C.

Sunday August 19th

Summary of Week 17

Week 17 (08/20 - 08/26/12)

Contents

Weekly Seminar

Monday August 20th

Tuesday August 21st

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for cloning in P. pastoris shuttle vector.

Wednesday August 22nd

  • Went to Dr. Joe Max Risse today and asked him about the mircoorganisms of the Fermentationgroup. He recommended Euglena gracilis, since it is without risk, big, colorful, and very healthy under the microscop.
  • I asked for the bacteria they have and he told me I could check if their Kocuria rosea is a wild type.
  • In the DMSZ catalog there are three strains of Kocuria rosea and all have been assessed to be of riskgroup 1, which is the safest group of bacteria and means it is unlikely that it will infect humans.
  • I also asked for Penicillium chrysogenum but the one they use is a GVO.
  • Ordered new primers for Xantomonas Campestris SDM, since there is still no positive colony and even gradient-PCR gave the wrong product


Thursday August 23rd

Friday August 24th

Saturday August 25th

STREET SCIENCE Visit of Danish guys till 08/27

Sunday August 26th

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) in front of the cellulose binding domain and the GFP. With a Freiburg-Prefix in front of the CBDs we could even easily switch directions of CBD and GFP.

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from the iGEM-plate into KRX and plated it on AMP-selection-dish.
Enzyme activity of ECOL, measured after incubation with 0.4 mM CuCl2 and measured at OD420 via oxidized ABTS.


Summary of Week 18

Week 18 (08/27 - 09/02/12)

Contents

Weekly Seminar

Summary of the weekend with our denish guests


Summary of StreetScience


Deadlines


Monday August 27th

Figure 1: Comparison of TVEL0 laccase stored in a refrigerator or in a freezer. Activity measurement was done by measuring oxidized ABTS via optical density 420.


Tuesday August 28th

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and digestion with SpeI and PstI
Figure 1: Activity measurements via oxidized ABTS and OD420 of ECOL, XCCL, TTHL, BHAL and BPUL in H2O. The enzymes were incubated in 0.4 mM CuCl2 before measurements.
Figure 2: Activity measurements via oxidized ABTS and OD420 of ECOL, XCCL, TTHL, BHAL and BPUL in H2O. The enzymes were not incubated in CuCl2 before measurements.
Figure 3: Activity measurements via oxidized ABTS and OD420 of ECOL, XCCL, TTHL, BHAL and BPUL in imidazole. The enzymes were incubated in 0.4 mM CuCl2 before measurements.
Figure 4: Activity measurements via oxidized ABTS and OD420 of ECOL, XCCL, TTHL, BHAL and BPUL in imidazole. The enzymes were not incubated in CuCl2 before measurements.

We sadly agree that rebuffering is necessary to ensure that our laccases are happy and active.

Wednesday August 29th

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.

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as template and the GFP_FW and GFP_His_compl primers. the most correct product was at 62°C.
Figure 2: SDS-PAGE of of purification from cultivation (08/28) of E.coli KRX with BBa_K863005, BBa_K863000, BBa_K863010, BBa_K863015 or BBa_K863020 with positive (BBa_K525710) and negative control (without plasmid). 250 mL in 1 L flasks without baffles, autoinduction medium with 60 µg mL-1 chloramphenicol at 37 °C for 12 hours. Purification of the supernatant via syringe method. The arrow shows ECOL (53 kDa).

Thursday August 30th

Friday August 31th

Figure 1: Activity measurements of two samples each of ECOL, XCCL, TTHL, BHAL and BPUL. KRX (cells without plasmid) and ligA (cells with a ligase A plasmid for the control of induction) have been used as a negative control. Enzymes have been incubated with 0.4 mM CuCl2 for 2h.
Figure 2: Activity measurements of two samples each of ECOL, XCCL, TTHL, BHAL and BPUL. KRX (cells without plasmid) and ligA (cells with a ligase A plasmid for the control of induction) have been used as a negative control.

Saturday September 1st

  • Plasmid isolations on the plated colonies and control restriction with NotI. Control digest showed for J23100 + J61101+ tthl_HIS, J23100 + J61101 + ecol_HIS positive bands. So we sent these plasmids for sequencing.

Sunday September 2nd

Summary of Week 19

Week 19 (09/03 - 09/09/12)

Contents

Weekly Seminar

Monday September 3rd

Figure 2: Activity in oxidizing ABTS of our produced BPUL laccase depending on time. Values are calculated by taking the average out of 4 measurements (n=4).

Tuesday September 4th


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-plasmid to express a cellulose binding domain (CBD) fused to a His-tagged GFP.

Wednesday September 5th

Figure 5: TVEL0 oxidizing different concentrations of ABTS over a time period of 5 minutes (n=4).

Thursday September 6th

Figure 6: SDS-PAGE of purification from our first fermentation of E. coli KRX containing BBa_K863000 (08/31). 3 L fermenter (Infors), 37 °C, pO2 of 50 % for 12 hours. Purification of the supernatant via Talon column. The arrow shows BPUL (58.6 kDa) in lane 4 and 5 (fraction 7 and 8).

Friday September 7th

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and  Status: 500

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in E. coli Rosetta-Gami 2.
Fragment Quantity [ng] Volume [µL]
5AOX1 26.5 0.44
MFalpha1 8.3 0.11
3AOX1 19.4 0.44
HIS4 73.0 2.16
pSB1C3 55.44 1.54
tAOX1 8.9 0,11

After incubation for 1 h at 50 °C, 2 µL were transformed in E. coli KRX cells by electroporation. And plated on selective LB Cm plates.

Saturday September 8th

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.

Fig. 7: BPUL activity of fractions 3 to 7 at 25°C with 0.01 mM ABTS (n=4).
Fig. 8: BPUL activity of fractions 15 to 19 at 25°C with 0.01 mM ABTS (n=4).


Fractions 6 and 7 show the highest activity leading to the assumption, that faction 7 contains as much protein as fraction 6 and that this amount is almost represented through BPUL laccase.


Fig. 9: Positive control represented through the activity of TVEL0. Negativ control with 0.5 mM CuCl in H2O (n=4).
Fig. 10: Comparison of the BPUL activity depending on CuCl incubation (n=4).
Fig. 11: Effects on BPUL by rebuffering from imidazole buffer in H2O (n=4).


Additionally we were interested in the impact on copper incubation. We have chosen fr. 18 and compared the activity of a copper incubated and a not copper incubated sample. With a incubation time of 2 hours with 0.5 mM CuCl2 we can double the activity of the BPUL laccase (see Fig. 10). Another interesting topic was the impact of imidazole buffer on the BPUL activity. For that we tested fr. 7 and fr. 18 in imidazole buffer and rebuffered in H2O. The results we got were unambiguously: BPUL in imidazole buffer didn't show much activity. Unfortunately we can't omit the rebuffering.




Sunday September 9th

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.

Figure 1: Activity measurements of wash and flowthrough samples of the TTHL purification. Measurements were done via OD420 and oxidized ABTS.

Summary of Week 20

Week 20 (09/10 - 09/16/12)

Contents

Weekly Seminar

Monday September 10th

Figure 1: SDS-PAGE of purification from fermentation (09/07) of E. coli KRX containing BBa_K863005, 6 L in NFL22, autoinduction medium, 37 °C, pO2 30 % for 12 hours. Purification of the supernatant via Ni-NTA column. The arrow shows ECOL (53 kDa).


Tuesday September 11th

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 and Status: 500

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and plated the ligation on AMP-selection-agar (because of the pSB1A2-backbone).

[[File:Bielefeld2012 ColiFr2-10 11 09.jpg|thumb|center|Figure 1: Activity measurements of ECOL fraction 2-10 after incubation with 0.4 mM CuCl2. Measurements were done via oxidized ABTS and at OD420.]

Wednesday September 12th

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-insert. Plated the positive colonies for plasmid-isolation.

Figure 2: SDS-PAGE of purification from fermentation (09/09) of E. coli KRX containing BBa_K863005, 3 L in Infors, autoinduction medium, 37 °C, pO2 30 % for 12 hours. Purification of the supernatant via Ni-NTA column.

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(09/09) behind a constitutive promoter.
Activity measurements of different fractions of ECOL. Measurements were done via oxidized ABTS and OD420.
Activity measurements of different fractions of TTHL. Measurements were done via oxidized ABTS and OD420.


Thursday September 13th

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-transformation clones

Figure 3: SDS-PAGE of purification from cultivation (09/09) of E. coli Rosetta-Gami 2 cells containing BBa_K863012 and Status: 500

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. 200 mL in flasks without baffles, LB medium, 60 µg/mL chloramphenicol and 300 µg/mL ampicillin, 37 °C for 48 hours. Purification of the supernatant via HisTrap column. The red arrow shows TTHL in lane 10-12 (elution 1-3).

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)








Activity measurements of ECOL regarding 100 mM sodium acetate buffer of different pHs. The enzymes were incubated with 0.4 mM CuCl2 before measurements. Activity was measured via OD420 and oxidized ABTS.
Activity measurements of ECOL regarding 100 mM sodium acetate buffer of different pHs without incubation with 0.4 mM CuCl2 before measurements. Activity was measured via OD420 and oxidized ABTS.

Friday September 14th

Figure 4: SDS-PAGE of purification from fermentation from (09/13) of E. coli KRX with BBa_K863000, 6 L in NFL22, autoinduction medium, 37 °C, pO2 50 % for 12 hours. Purification of the supernatant via Ni-NTA column. The red arrow shows BPUL







Anthracen control
Ethinylestradiol control

We analyze Anthracen and Ethinyl estradiol from the 13th September. As you can see in the following figures Anthracen distingrates and Ethinyl estradiol on the other hand was stable. The corelation between the first peak and the other two peaks on Ethinyl estradiol may a pipetting mistake since the other two peaks are nearly simular. Distingration on Anthracen, a polycyclic aromatic hydrocarbon, were tested in different media and we determine that it depends on the Britton-Puffer (read more)

Saturday September 15th

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to have pSB1C3 backbone for the last clonings.

Sunday September 16th


Summary of Week 21

Week 21 (09/17 - 09/23/12)

Contents


Weekly Seminar


Monday September 17th

Figure 1: Activity of ECOL measured via oxidized ABTS at OD420 in relation to a MeOH gradient.
Figure 2: Activity of ECOL measured via oxidized ABTS at OD530 in relation to a MeOH gradient.
Figure 3: Activity of ECOL measured via oxidized ABTS at OD420 in relation to a acetonitrile gradient.
Figure 4: Activity of TTHL measured via oxidized ABTS at OD420, showing activity in each fraction after prufication.
Figure 5: Activity of ECOL measured via oxidized ABTS at OD530in relation to a acetonitrile gradient.
Figure 6: Activity of BHAL measured via oxidized ABTS at OD530, showing activity in each fraction after prufication.

Tuesday September 18th

The shuttle vector was linearized with EcoRI and SpeI and was transformed in competened P. pastoris cells.

Fig. 1: Measurements of ECOL in a CuCl gradient of 0,1 to 0,7 mM. The activity was measured via oxidized ABTS at OD420, (n=4).
Fig. 2: Activity measurements of TTHL via OD420.








Wednesday September 19th

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and CBDclos_Freiburg with GFP_Freiburg and transformed it into KRX.
BPUL laccase activity measured via ABTS and OD420 under usage of different pHs of sodium actate buffer. The results show a high activity at pH 5.
Negative control for pH measurements using ABTS. ABTS is oxidized through havy metals like Cu, which we are using for laccase activity tests. At different tested pHs ABTS gets oxidized but not very fast allowing us to leave it outer consideration.
BPUL laccase activity measured with different concentration of CuCl showing that an incubation with 0.6 mM CuCl is optimal for the laccae activity, but higher CuCl concentrations seem to interfere in the activity.
Negative control for analyzing the oxidization of ABTS by different CuCl concentrations. An effect on the oxidization of ABTS is clearly distinguishable, but between the applied concentrations there is no difference in OD420.
Characterization of the activity of BPUL with sodium acetate buffer and Briton-Robinson Buffer at pH 5. Using the Briton-Robinson Buffer the saturation of BPUL activity is reached slower but stays constant. With sodium acetate buffer the maximum is reached considerably earlier.
BPUL activity under different concentrations of acetonitrile. Acetonitrile has a small impact on the activity but indicates a decrease in activity the more actetonitrile is used.
BPUL activity under different concentrations of MeOH. MeOH seems to have no impact on the activity using this small amounts of it.



Thursday September 20th

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arrived. We did PCR on GFP with the new primers for cloning in our shuttle vector.
Temperatur measurements of ECOL at 10°C and 25°C. Measurements were done via optical density 420 nm.
Temperatur measurements of BPUL at 10°C and 25°C. Measurements were done via optical density 420 nm.

Friday September 21st

P. pastoris colonies are seen and are picked into liquid culture for genomic DNA isolation and genotype characterisation.

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and GFP PCR product with AarI and ligation. Transformation in E. coli KRX cells.

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with CBDclos+GFP again and plated on the right selection-agar this time!
ECOL activity with different concentration of ABTS.
BPUL activity with different concentration of ABTS.

Saturday September 22nd

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, with Status: 500 Content-type: text/html

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and without plasmid.

Sunday September 23rd

genomic DNA could be isolated and have to be analyzed with primers: 5AOX-Genotyp-FW and TT-Genotyp-RV.

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with S3N10_Cex_compl- and S3N10_GFP-primermix to generate a 13 amino acid-linker between the cellulose binding domain and the GFP.
Figure 1: Growth kinetics of flask cultivation of E. coli KRX with Status: 500

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and without plasmid as negative control using LB-medium and of E. coli KRX with Status: 500

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using autoinduction medium. Further parameters: final volume of 60 mL, 60 µg/mL chloramphenicol, 37 °C, durance: 12 hours.

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and E. coli KRX without plasmid as negative control

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for comparison.
Figure 2: SDS-PAGE of purification from our first fermentation of E. coli KRX with BBa_K863012 (09/15). 6 L in fermenter NFL22, 37 °C, pO2 of 30 % for 12 hours., LB-medium with 60 µg/mL chloramphenicol and 300 µg/mL ampicillin. Purification of the supernatant via His-trap column. The red arrow shows TTHL in lane 9-11 (elution 1-3).

Summary of Week 22

Week 22 (09/24 - 09/30/12)

Contents

Weekly Seminar

Monday September 24th

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, E. coli KRX without plasmid (negative control) and with Status: 500

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(positive control). 60 mL in 250 mL flasks without baffles,LB-medium or autoinduction medium for Status: 500

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, 60 µg/mL chloramphenicol, 37 °C for 12 hours.

















Tuesday September 25th

Wednesday September 26th

Thursday September 27th

Friday September 28th

Saturday September 29th

Sunday September 30th

Summary of Week 23

Week 23 (10/01 - 10/07/12)

Weekly Seminar

Monday October 01st

Tuesday October 02nd

Wednesday October 03rd

Thursday October 04th

Friday October 05th

Saturday October 06th

Sunday October 07th


Summary of Week 24

Week 24 (10/08 - 10/14/12

Contents


Monday October 08th

All: Day off in Amsterdam!!!

Tuesday October 09th

Wednesday October 10th

Thursday October 11th

Bioengineering NFL 19L fermenter, autoinduction HSG medium, 60 µg/mL chloramphenicol, 19 L, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 12 NL/m, 16 hours. HSG medium was choosen to get a high biomass concentration with hope for a higher amount of laccases.

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(09/09) behind a constitutive promoter. (500mL preculture)

Friday October 12th

Figure 1: Fermentation of E. coli KRX with Status: 500

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(ECOL) in an Bioengineering NLF 19, scale: 12 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold pO2 at 50 %, OD600 measured every 30 minutes.
Figure 2: Fermentation of E. coli KRX with Status: 500

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(BPUL) in an Bioengineering NLF 19, scale: 12 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold pO2 at 50 %, OD600 measured every 30 minutes.


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(09/09)

Bioengineering NFL 19L fermenter, HSG medium, 60 µg/mL chloramphenicol, 300 µg/mL ampicillin, 12 L, 37 °C, stirrer on cascade to hold a pO2 of 50 %, airflow: 12 NL/m, 22-24 hours. HSG medium was chosen to get a high biomass concentration with hope for a higher amount of laccases.

Saturday October 13th

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(09/09) were harvested and stored at 4°C until purification.
Figure 1: Fermentation of E. coli KRX with Status: 500

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(ECOL) in an Bioengineering NLF 19, scale: 12 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold pO2 at 50 %, OD600 measured every 30 minutes.
Figure 2: Fermentation of E. coli KRX with Status: 500

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(BPUL) in an Bioengineering NLF 19, scale: 12 L, autoinduction medium + 60 µg/mL chloramphenicol, 37 °C, pH 7, agitation on cascade to hold pO2 at 50 %, OD600 measured every 30 minutes.



Sunday October 14th


Summary of Week 25

Week 25 (10/15 - 10/21/12)

Contents

Monday October 15th

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(09/09) were disrupted via high-pressure homogenizer and filtrated by Millipore Pellicon XL 50 with first 300 kDa to seperate all celldebris and 10 kDa to concentrate the laccases. The concentrated solutions were purified by the Ni-NTA-column.

Tuesday October 16th

Bielefeld2012 Prot vor Umpuffern.jpg

Taking samples for immobilization time analyzation.

Wednesday October 17th

Bielefeld2012 Prot nach Umpuffern.jpg

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).

Figure 1: SDS-Page of purification from the 12 L fermentations from 10/11 (BBa_K863000, BBa_K863005) and 10/12 (BBa_K863012, Status: 500

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). Purification of the supernatant via His-trap column, step gradient with 5 %, 50 % and 100 % elution buffer.

Measurement of Protein concentration with Roti-Nanoquant and analyses of binding capacity.

Thursday October 18th

Activity assay of each purified fraction of our new cultivation with ECOL. Samples were re-buffered into H2O and the protein amount in each fraction has been adjusted. The Measurement was done using the standard activity assay protocol over night.
Activity assay of each purified fraction of our new cultivation with BPUL. Samples were re-buffered into H2O and the protein amount in each fraction has been adjusted. The Measurement was done using the standard activity assay protocol over night.
Activity assay of each purified fraction of our new cultivation with BHAL. Samples were re-buffered into H2O and the protein amount in each fraction has been adjusted. The Measurement was done using the standard activity assay protocol over night.
Activity assay of each purified fraction of our new cultivation with TTHL. Samples were re-buffered into H2O and the protein amount in each fraction has been adjusted. The Measurement was done using the standard activity assay protocol over night.







































Friday October 19th

Slope of the two most active fractions of purified TTHL laccase. By comparing them, the amount of contained laccase in the fractions had been calculated.


Saturday October 20th

Activity assay of our new ECOL laccase with different ABTS concentration to find the substrate saturation of ECOL. 616 ng ECOL laccase (incubated with 0.4 mM CuCl2) and 20 % Britton-Robinson buffer (pH 5) were applied.
Activity assay of our new BPUL laccase with different ABTS concentration to find the substrate saturation of BPUL. 616 ng BPUL laccase (incubated with 0.4 mM CuCl2) and 20 % Britton-Robinson buffer (pH 5) were applied.


Sunday October 21st

Genomic isolation was done with the Promega Wizard genomic DNA purification system kit of our approximately 50 yeast clones with integrated (a) GFP and (b) tvel5.

Activity assay of our new BHAL laccase with different ABTS concentration to find the substrate saturation of BHAL. 616 ng ECOL laccase (incubated with 0.4 mM CuCl2 and 20 % Britton-Robinson buffer pH 5) were applied.
Activity assay of our new TTHL laccase with different ABTS concentration to find the substrate saturation of TTHL. 616 ng TTHL laccase (incubated with 0.4 mM CuCl2 and 20 % Britton-Robinson buffer pH 5) were applied.



















Summary of Week 26

Week 26 (10/22 - 10/28/12)

Contents

Monday October 22nd

The identification of positive yeast clones and determination of the phenotype (M+ or MS) was done by PCR with the primers 5AOX-Phenotype-FW and TT-Phenotype-RV. The result of the PCR was: no GFP integrated clones and three tvel5 integrated (1.9 kb and 2.2 kb) clones. The cassette, including tvel5 and his4, was recombinated by a single cross over, because the 2.2 kb PCR product of the aox1 gene has been formed. Minimal methanol medium was inoculated with these three positive clones with the M+ phenotype.

BBa_K863204 was used as a controll for PCR with the primer 5AOX-Phenotype-FW and TT-Phenotype-RV. Expected lane: 437 bp.
PCR product of the gDNA from the culture 1, 2 and 3 (C1, C2, C3) with the primer 5AOX-Phenotype-FW and TT-Phenotype-RV. Expected lane: 1.9 kb (tvel5) and 2.2 kb (aox1).



























BHAL activity measured under different pH conditions (pH 4 to pH 9) to determine the pH optimum. 616 ng Laccase and 5 mM ABTS were used.
TTHL activity measured under different pH conditions (pH 4 to pH 9) to determine the pH optimum. 616 ng Laccase and 5 mM ABTS were used.
ECOL activity measured under different pH conditions (pH 4 to pH 9) to determine the pH optimum. 616 ng Laccase and 5 mM ABTS were used.
BPUL activity measured under different pH conditions (pH 4 to pH 9) to determine the pH optimum. 616 ng Laccase and 5 mM ABTS were used.








































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in Status: 500

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showed a deletion within the ORF of the GFP; this seemed to be the reason that the colonies are not fluorescent.

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with GFP_Freiburg Pre- and Suffix Primers

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with CBDcex_Freiburg and GFP_Freiburg_compl Primers

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with CBDclos_Freiburg and GFP_Freiburg_compl Primers

Tuesday October 23rd

Bielefeld2012 ECOL hoch.jpg
Bielefeld2012 BPUL hoch.jpg


Bielefeld2012 BHAL ABTS hoch.jpg
Bielefeld2012 TTHL hoch.jpg


Wednesday October 24th

Thursday October 25th


Last offline analysis of the the two cultivations of E.coli KRX containing BBa_K863000and BBa_K863005 with Carobon Source Detection with HPLC.

Thursday October 25th

Activity test of cultivation (culture 1, 2, 3) supernatant of BBa_K863207. The control is the supernatant of P. pastoris GS115.


Friday October 26th

Final offline analysis of the E.coli Rosetta Gami 2 cultivations with BBa_K863012(09/09) and Status: 500 Content-type: text/html

Software error:

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(09/09).

Saturday October 27th

Sunday October 28th

Promega logo 180x109.gif Logo merck.jpg BioCircle.JPG Bielefeld2012 Evonik.jpg Bielefeld2012 Baxter.png Logo knauer.jpg Logo iit.jpg Bielefeld2012 BIEKUBA.jpg Logo biometra.jpg Logo bio-nrw.png Bielefeld2012 Logo ERASynbio.jpg

Retrieved from "http://2012.igem.org/Team:Bielefeld-Germany/Labjournal"