December
Wet Lab work has not yet started.
Dry Lab work has not yet started.
15/12/15:
- Organization:
We had our first team meeting and started with introducing us to each other. To get an overview over our background, every team member shortly described their bachelor topics. We also had a discussion on time requirements decided that team members should only take one core module during the summer term. Furthermore, it was suggested to organize lab work in shifts. We also exchanged first ideas for possible projects and organized a meeting with Professor Stülke who wanted to present a possible project.
15/12/21:
- Organization:
Professor Stülke and members of his lab shortly introduced a possible project from his department based on the idea of a minimal organism. After the meeting, the team members discussed the suggestion. We decided to ask other professors for different project ideas, before choosing a topic.
January
Wet Lab work has not yet started.
Katzen liegen nicht faul rum. Sie verschönern den Raum.
16/01/08:
- Organization:
Several team members introduced additional project ideas based on former projects and own ideas. Tobias S. agreed on asking Professor Daniel, who supervised the iGEM team from 2015, for a different project.
16/01/12:
- Organization:
Tobias S. introduced the project proposed by Professor Daniel. The project would aim at constructing a synthetic Vitamin B12 exporter and testing it in different production organisms. We also discussed advantages of this project idea compared to others.
16/01/13:
- Organization:
We decided on the project “Vitamin B12 exporter” proposed by Professor Daniel. All team members voted in favor of the project.
16/01/18:
- Organization:
We had the first team meeting with our supervisors discussing a rough timeline with important deadlines.
We also agreed on collecting title ideas and searching literature for Vitamin B12 binding proteins, Vitamin B12 dependent reactions and production organisms for the next meeting. - Financials:
We started planning the project funding by sponsors and internal university funding.
16/01/21:
- Organization:
After having collected many different project titles during a very funny meeting, the majority of our team voted for “B12 Synporter- Another brick in the wall”.
February
Wet Lab work has not yet started.
Katzen liegen nicht faul rum. Sie verschönern den Raum.
16/02/01:
- Financials:
We decided to design and print a booklet on our project to send it to possible sponsors. Amongst others, information on the iGEM competition, the background to our project, the team and sponsoring suggestions should be included. The topics were divided between team members. The organization team agreed on writing the email text and the text for a letter to address sponsors.
March
Wet Lab work has not yet started.
Katzen liegen nicht faul rum. Sie verschönern den Raum.
16/03/03:
- Dry lab:
Together with the supervisors, we decided on the three production organisms typhimurium TA100, R. planticola and S. blattae to test the constructs for Vitamin B12 exporters. We did not yet choose different B12 binding proteins, which can be used in the exporter. Further literature research is needed for that. However, we already discussed possible experiments to test the B12 productivity in the production organisms in the end. Those include a photometric, a biological and an antibody assay. The strains for our production organisms have to be ordered from DSMZ. For a biological assay we also have to search for a B12 auxotrophic strain and a B12-free medium.
16/03/18:
- Financials:
We registered the team for the IDT website to use their sponsoring for iGEM teams to synthesize our constructs.
16/03/29:
- Dry lab:
After consulting our supervisors, we decided on three different constructs (GlmS, BtuF and MutB) to design our synthetic exporter.
April
Week 1: GH, TS, TK
- Antibiotic resistance:
After having received our production strains S. blattae, R. planticola and S. typhimurium TA 100 we could start lab work with testing them for natural antibiotic resistances. For this, they were grown in culture overnight and plated on LB-Agar plates supplemented with different antibiotics.
Week 2: TK, GH, LK, IF, MS, TS
- Antibiotic resistance:
Due to inconsistent results in the week before, the test for antibiotic resistances had to be repeated. We additionally tested for resistance against chloramphenicol and used more different concentrations of the antibiotics and dilutions of the pre-cultures. The results showed, that all production strains have no antibiotic resistance for kanamycin. It can therefore be used as antibiotic resistance on our plasmids. - Bacterial identification:
To check the identification of our production strains, we performed a 16S rRNA PCR. All strains were correct, so glycerol stocks could be prepared. - Competent cells:
For the following experiments, we had to prepare electro competent cells of our productions strains and E. coli DH5α. This was done first for E. coli DH5α and S. typhimurium TA100 according to protocol.
Week 3: IF, NE, ME, MS, TK
- Competent cells:
Electrocompetent cells for S. blattae and R. planticola were prepared.
Week 4: IF, LK
- Test transformation:
To check if our prepared electro competent cells were indeed electro competent, we transformed them with a simple vector containing a kanamycin resistance according to protocol and grew them on plates over night. Unfortunately, only E. coli DH5α showed resistance against kanamycin, suggesting that the transformation or the preparation of competent cells was not successful for the others.
Katzen liegen nicht faul rum. Sie verschönern den Raum.
16/04/01:
- Wet lab:
We planned the first steps, which needed to be taken in the lab. We have to grow the strains, make the cells electro competent, proof their identity via 16S PCR and check for possible antibiotic resistances. - Dry lab:
More research is needed on the TAT secretion system and possible export signals, as well as possible vector systems. It was decided that they should be inducible by arabinose, suggesting the pBAD vector system.
16/04/11:
- Wet lab:
After discussing the first results from the lab concerning the antibiotic resistances, it was decided to repeat the experiments with chloramphenicol as additional antibiotic and using different concentrations.
For the following weaks, the team is devided into three groups, one testing antibiotic resistance again, one starting to prepare electro competent cells and the third team designing the constructs. - Organization:
We agreed on having a regular iGEM meeting with the supervisors every two weeks on Monday, 11 am. Furthermore, the organization team will start writing guide lines for lab work and Ines will organize a log book for signing in lab working hours.
16/04/25:
- Wet lab:
The electro competence of our cells will be tested with a simple kanamycin vector.
To dissolve the synthesized constructs from IDT, we will use either a 10 mM Tris Buffer or an elution buffer from a purification Kit before making aliquots. - Dry Lab:
If we also want to use denitrificans as a production organism, we need to search for the specific name and ask Ute, if the strain is available in the lab.
Tobias S. is in contact with Thermo Fisher for our order of the pBAD202 D-TOPO vector. - Financials:
The sponsoring by B. Braun was confirmed. We can also start sending letters to possible sponsors because the etiquettes for the envelopes are prepared. - Organization:
We might try to find a bachelor student, who wants to join our project after the “Bio & Brezeln” presentation. They would need to send a letter with a written application to the “Prüfungskommission” to get credits for participating. - Human practice:
The “Bio & Brezeln” presentation will be held by Tobias S., Tobias K. and Miriam.
Miriam will also contact the “Göttinger Tageblatt” and ask them to write an article about our participation in the iGEM competition. - Events:
We intend to participate in the meetup of German iGEM teams in Marburd. Tobias S. will organize the registration. Furthermore, we want to present our team at the “Göttinger Altstadtlauf” and the sports competition “DIES” from our university.
May
Week 1:
- No wet lab work done.
Week 2: IF, LK, NE, TS, GH
- Test transformation:
The previously prepared cells of our production strains were again tested for electro competence. The protocol was slightly changed using SOC medium instead of LB-medium and incubating the bacteria at their temperature optima (R. planticola: 30 °C, others: 37 °C). However, the cells again did not grow, showing that they were not electro competent.
Week 3: NE, TK, GH
- Preparation:
For the following week, large amounts of media had to be prepared.
Week 4: NE, GH, MS, AB
- Competent cells:
The preparation of competent cells was repeated for S. blattae, R. planticola and S. typhimurium TA 100. The cells were again incubated at their respective temperature maxima.
Katzen liegen nicht faul rum. Sie verschönern den Raum.
16/05/09:
- Wet lab:
The transformations need to be repeated, because they did not work. We might need to adjust the protocol.
Additionally, the competent cells need to be checked for electro competence. - Dry lab:
We will use snapgene to design our vectors and primers. - Human practice:
For the Master Information Day at our university we need to prepare a poster to present our project and the iGEM competition to future master students.
Furthermore, we need to work on the presentation for the “Bio & Brezeln” event at the 26th of May. - Financials:
We were sponsored by Zymo Research with Kits and T-Shirts. To thank them, we will take a group picture with the shirts and post it on our facebook profile. - Events:
We decided to participate in the meeting of the European iGEM teams in Paris (16/06/01-16/06/03) and intend to book the hotel rooms organized by the iGEM teams from Paris and bus tickets.
16/05/18:
- Wet lab:
The vector from Thermo Fisher has arrived, so we could start with inserting our constructs. The work on electro competent cells, test transformations and ligation of the MutB construct should be finished by 6th of june. - Human practice:
We intend to present our project and iGEM at the “GZMB Sommerfest”. In return, we will offer our support in organizing the event. - Organization:
We discussed the final design for our team T-Shirts and possible offers for printing them.
16/05/30:
- Dry lab:
Genis will help us to design the first primers and give us an introduction into the snapgene program. - Wet lab:
The newly prepared electro competent cells need to be tested. If necessary, we will repeat the preparation. - Human practice:
We need to continue working on our poster for the Master Information Day. - Organization:
For our website, we want to create a new figure of our “Synporter”.
June
Week 1: IF, LK, TK, GH, ME, AB
- Test transformation:
The newly prepared electro competent cells were tested and compared with a test transformation of cells provided by our supervisor Genis. Only the S. blattae cells showed antibiotic resistance.
- MutB construct:
After having received the synthesized constructs from IDT, we could start to prepare the constructs. MutB had to be ordered with a restriction site and synthesized in two fragments due to its length. Therefore, it had to be digested with HindIII, purified and ligated.
Week 2:
- No wet lab work done.
Week 3: AB, ME
- Competent cells:
The preparation of electro competent cells was repeated for R. planticola and S. typhimurium TA 100 and test transformations with a simple kanamycin vector were successful.
- MutB construct:
The ligation of the two fragments was repeated. Additionally, the synthesized MutB1 and MutB2 fragments were amplified by PCR, followed by gel electrophoresis and gel extraction to prepare a stock.
Week 4: AB, ME
- MutB construct:
The amplified MutB fragments were digested with HindIII, purified and ligated. However, gel electrophoresis showed that none of the ligations worked yielding products of wrong sizes.
Katzen liegen nicht faul rum. Sie verschönern den Raum.
July
Week 1: AB, ME
- MutB construct:
Restriction, purification, ligation and gel electrophoresis had to be repeated, but the results were again negative.
- GlmS and BtuF constructs:
The synthesized constructs were cloned into the pBAD202 D-TOPO vector. In contrast to the suggested protocol, we used only half of the amount of the vector. The transformation was performed according to protocol into TOP10 E. coli. The resulting colonies were transferred into liquid culture and incubated over night. From the grown cultures, plates for storage were prepared and plasmid preparations were done.
Week 2: AB, ME
- MutB construct:
Due to the difficulties of ligating the two fragments of MutB, we tried a different approach and prepared fragments with overhangs by PCR using newly ordered primers. After gel electrophoresis and purification, the fragments were directly ligated.
- BtuF construct:
To check if BtuF was correctly inserted into pBAD202 it was digested with SmaI. The gel after electrophoresis did not show any bands.
Week 3: ME, TS, TK, MS
- MutB construct:
To find out if the ligation was successful, a PCR to recover the fragments was done with the respective primers.
- GlmS construct:
To check if GlmS was correctly inserted into pBAD202 it was digested with AccI. The gel after electrophoresis did not show any bands.
Week 4: TS, TK, MS, AB
Week 5: TS, TK, MS, AB
Katzen liegen nicht faul rum. Sie verschönern den Raum.
16/07/06:
- Dry Lab:
Our binding proteins might only be partly loaded. We could check this quantitatively through Western blot using the epitope tag or the His-Tag. - Giant Jamboree:
We started looking for flights to and from Boston and discussed hotel options. Everyone has to do the ESTA registration to be allowed to travel to the USA. - Financials:
At the beginning of August we will check how much money is available for the Giant Jamboree. - Human practice:
We want to participate at the “GZMB Sommerfest” and will contact the responsible persons. Additionally, we will try to develop a concept for a school project and might organize interviews with experts. - Cooperation:
The iGEM team Leuven is working on an interesting project and we might be able to cooperate with them.
August
Week 1: TK, AB, MS, GH, ME
Week 2: ME, MS, GH, AB, TK
Week 3: AB, TK, GH, MS, TS, LK
Week 4: GH, LK, ME, TK, TS, IF, MS, AB
Katzen liegen nicht faul rum. Sie verschönern den Raum.
16/08/08:
- Events:
The meetup of the German iGEM teams in Marburg was a good experience and successful. We might cooperate with Bielefeld (on aptamers) and Darmstadt (on biosafety). - Wet lab:
Next steps are the transformation of BtuF and GlmS and sequencing afterwards to confirm the correct orientation. We then will continue with expression tests.
For MutB the new primers with overhangs have arrived for the new approach of ligating the two fragments. - Giant Jamboree:
The team members need to register for the Giant Jamboree in Boston until the 31st of August. Our planned travel time is from 16/10/25 to 16/11/02.
16/08/22:
- Wet lab:
GlmS and BtuF have to amplified with the new primers and sequenced. In addition to that new plated need to be prepared.
Genis pointed out that the primers in MutB might overlap to much. - Giant Jamboree:
The registration fee has to be paid by the team members for now, but we might get an advance of travel costs. We also discussed options for flights and hotels and talked about organizational things. - Financials:
We will try to contact Sartorius again and ask them for sponsoring.
September
Week 1: AB, ME, MS, GH, LK
Week 2: LK, GH, AB, TK, ME
Week 3: GH, LK, IF, MS
Week 4: GH, LK, IF, ME, TS, AB, MS
Katzen liegen nicht faul rum. Sie verschönern den Raum.
16/09/05:
- Wet lab:
In the following weeks, lab work needs to be divided into different teams.
MutB and GlmS were successfully subcloned and can be transformed into the arabiniose negative coli strain for expression experiments.
It also needs to be tested if the thioredoxin fusion protein in the pBAD202 vector influences the expression of our symporter protein. - Financials:
We talked about the travelling costs for Boston and are happy, that we get supported by Sartorius as new sponsor. - Human practice:
The postcards from the Human practice action initiated by the iGEM team Düsseldorf have to be distributed in public.
We also will present our project results in a colloquia at our institute at the beginning of the next semester.
16/09/21:
- Wet lab:
The B12 detection tests have to be started. The strains will be cultivated according to protocol. For the tests minimal medium has to be prepared and the periplasmic space will be extracted by ultracentrifugation.
For GlmS the constructs without thioredoxin need to be checked by repeating the SDS-PAGE of the expression experiments. - Financials:
The travel expense accounting for Boston was done. - Human practice:
For the Wiki, texts about our human practices need to be written.
In addition to that, we might contact the schools again, organize a “lab work” event for students and arrange a BBQ for the new master students.
October
Week 1: GH, LK, IF, ME, TS, AB, MS
Week 2: IF, ME, LK, GH, AB, MS, TK
Week 3: GH, LK, MS, TS, AB, ME, IF, TS
Week 4:
Katzen liegen nicht faul rum. Sie verschönern den Raum.
16/10/11:
- Giant Jamboree:
We met to plan our presentation for the Giant Jamboree. After swapping ideas, we came to an agreement and are now on our way to prepare the presentation.
Furthermore, we talked about the dress code for the day of our presentation and some further organizational stuff according our trip to Boston.
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