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Team:Potsdam Bioware/Lab/Group Meetings
From 2012.igem.org
Group Meetings
Summary 2012-03-12
presentation of topics, progress and deepening of projects
Attendees: Christopher, Tom S., Rico, Kevin, Kerstin, Sascha, Mario, Tom O.
- magnetosome-beads: Christopher
idea: functionalization of magnetic beads with proteins display method with magnetic beads:
- knock-out mutant for magnetosomes – biosynthesis and transformation with essential gene
- for protein interaction, transcribtion of gene and biosynthesis of magnetosomes
- purification with magnetic force
problems & to do: which stains are suitable? ‘’Magnetospirillum WT‘‘ or ‘‘E.coli‘‘ transformation?
- hypermutated antibody by transfection: Rico
idea: improvement of antibody affinity through integrated deaminase which leads to hypermutation
- cell line with antibody + Fc-receptor for antibody presentation on surface
- virus with antigen on envelope that can be bind specifically by antibody and releases incorpo-rated deaminase gene into cell
- hypermutation
- test for affinity
problems & to do: mechanism of hypermutation? CHO cell line (able to hypermutate sequences?) or myeloma cells?
- voltage responding proteins: Sascha
idea: linkage of proteins to voltage sensitive transmembrane domains
- application for voltotaxis via PI3-kinase for example
problems & to do: stability without membrane connection?
Next meeting: Wednesday 4th April at 5 pm, B0.01 H25!
Summary 2012-04-04
Deepening of project ideas Attendees: Christopher, Tom S., Rico, Kevin, Kerstin, Sascha, Tom O. Presentation of project by Rico
- hypermutation by protein E2 of Hepatitis C Virus
- protein E2 leads to hypermutation in B-cells by activation of AID through CD81 (activation in-duced Deaminase)
3 concepts:
- myeloma cells with antibody on surface. Addition of E2 and virus with antigen on surface + siR-NA against AID – reduction of AID activity after virus binding
- myeloma cells that secrete antibody, addition of E2 and antigen on surface - saturation of virus by improved antibody
- myeloma cells that secrete antibody and AID knock-out, addition of virus with AID on surface and E2 – induction of hypermutations through binding
(E2 with linker peptide and antigen) to do: are there any publications that describe E2? Is the mechanism of action well known? Why is CD81 important and which cells lines are robust enough to survive the pathway? How can you control CD81? How long does the hypermutation take? How is it possible to select cells? idea: patent, peptide linker for BioBrick, DNA-Origami as linker? magnetosomes
- talk to people from the MPI, exists any application for the project mentioned above?
Next meeting: Friday, 13th April, 8 am (Vorlesungszeitraum)
Summary 2012-04-20
Attendees: Tom S., Rico S., Tom O., Kevin S., Mario K., Tobias P., Kerstin W., Xenia A., Barbara M., Katharina T., Stefan G., Maria K., Tarek S., Christopher K., Sascha L., Laura R. 1. reminder: all team members have to register for wiki! 2. presentation of wiki page and editing of wiki (Tom) Presentation was uploaded! 3. deepening of the project
- A. magnetosomes (Christopher): idea was rejected, preparation/creation of knock-out mutants would have been too time consuming and can thus not be realized and additionally a lot of genetic know-how would have been needed
- B. hypermutation by protein E2 of Hepatitis C Virus (Rico)
protein E2 leads to hypermutation in B-cells by activation of AID via CD81 (activation induced Deaminase) idea und goal of the project:
- increasing antibody -activity and specificity + adjustable hypermutation by AID
- binding of virus-antigen to antibody on surface of beads have to be selected
- cells to be used: CHO
possible stages of the project:
- induction of hypermutation
- transfection of cells with antibody
- transfection of AID + detection/proof of hypermutation
- binding of virus particles to antibody on surface
- Establishment of selection system
Which steps can be realized during the project?
- antibody on cell surface?
- can virus bind?
to do:
- possible usage of camellid antibody?
- is a switchable expression of surface-antibody by Tet on/off- or Cre-Lox-system possible?
- C. Adeno-assoziated Virus = AAV (Tarek)
idea:
- using AAV as vector, transforming transgenes (GOI) between ITRs (inverted terminal repeats) of AAV
- multiplication of transgenes would be possible via helper plasmid and not non-pathogenic AAV helper plasmids
Advantage: good purification possible, expression can be controlled
Disadvantage: expression "without help" slowly, possible damage of genes by undirected integra-tion
Next meeting: Friday 27th of April 8:00 am (Vorlesungszeitraum)
Following tasks have to be prepared:
- iGEM-judging criteria, creating time table (Kevin)
- iGEM-requirements for cloning and Biobricks (Maria & Laura)
- Sponsoring (Kathi, Mario & Tom)
- topical focus:
- How to design the virus construct? (Tobias, Xenia und Tarek)
- Transfection von camellid-AK (Sascha)
- AID-specificity (Rico, Chris) Chris will start with the following paper [http://www.sciencedirect.com/science/article/pii/B9780123942807000051 Paper] or already known?
- Tet-on/Tett-off- und Cre-Lox-systeme for switchable expression (Tom & Stefan)
--> to Rico and Tarek: could you maybe upload your presentation from the 20th April?
Summary 2012-04-12
Attendees: Mario K., Laura R., Tobias P., Christopher K., Kerstin W., Maria K., Stefan G., Rico S., Tarek S. Xenia A., Katharina T., Sascha L. Tom O., Tom S., Kevin S., 1. Presentation of iGEM in general for new group members: Kevin
- information about judging criteria are necessary!
2. presentation of Biobricks in general and of already existing parts: Maria 3. sponsoring:
- a sponsoring letter should be reviewed for a second time and should be normed to one page, as soon as it is ready we will have to talk about it again; application for financial help for iGEM pro-ject ( Kristian)
4. presentation of iGEM 2010 Freiburg Bioware for possible virus construction: Tobias. 5. discussion of transfection:
- stable transfection is time consuming and complicated, CHO-cells should have integrated the Flp- ( Invitrogen with possible concession?), or with contact to anyone? Further information will have to be discussed at the next meeting.
6. presentation of Tet on Tet off systems by Stefan. 7. presentation of time table from Thursday by Maria.
- separation into groups with different partial projects:
- Judging: Kevin
- CHO: Tom O., Sascha, Tarek, Stefan, Maria, Kerstin
- AID: Rico, Tom S., Chris, Mario, Basia
- Virus: Laura, Tobias, Kathi, Xenia
- design of logo: Kathi
- Sponsoring: Tom S., Mario, Kathi
8. iGEM e.V.
- Kristian wrote the membership bid for iGEM e.V., members will have to sign
- advantages of iGEM e.V.: receipts can be easily issued via the society. Furthermore the collec-tion of donations can be handled more easily.
Next meeting: Wednesday, 02th May 6 pm
Summary 2012-05-04
Attendees: Tom, Rico, Tom O., Kevin, Stefan, Basia, Tarek, Kathi, Maria, Xenia, Christopher, Tobias, Laura, Sascha, Mario, Kerstin Protocol: Kevin Moderation: Kerstin 1. Discussion of time tables by Basia, groups are up to date 2. Presentation by Kathi: logo
- 4 logos can be choosen
- more logos will be uploaded for a further discussion/voting
3. Presentation by Xenia: AAV2-virus (Freiburg iGEM 2010)
- presenting the way of function
- alternatives in plasmids (3plasmids)
- how to create the plasmids
- transfection of target cells
- possible purification approaches
4. progress report by antibody group
- desired camellid antibody not possible
- alternative: mouse -> problem: heavy and light chain
- quest for an antibody with low affinity, for mutation by AID that can possibly leads to a better affinity mouse: c-Myc antibody single domain antibody: lysozyme further problems: is there a convenient method to measure affinity with cells? 5. presentation by Sascha: Flp-in system
- way of function of the system (plasmids)
- prize and needed material
- transfection method: advantage and disadvantage
6. presentation by Rico: AID
- AID-transfection + vector
- strategy and proceeding
- selection method
- process and material
7. collecting ideas for sponsoring
- for donations from a different field than biology, arguments have to be found to make our topic more interesting
Next meeting: Tuesday 11th May
Summary 2012-05-11
Attendees: Tom, Rico, Tom O., Kevin, Stefan, Basia, Tarek, Kathi, Maria, Xenia, Christopher, Tobias, Laura, Sascha, Mario, Kerstin
- CHO cells arrived!!!
- presentation and comparison of possible antibodies
- anti-c-Myc or anti-lysozyme
- acquisition of antibody not clear yet
- c-myc antibody from project group Braunschweig
- further ideas?
- which size of antibody/ Fab fragment is eligible for Flp-in system
- how stable is the system?
- transmembrane domain?
- is the antibody compatible with the Flip-in system
- measuring the affinity by fluorescence polarizing
- presentation by Christopher of expression vectors
- decision for logo 2!
Next meeting: Tuesday 15th May 6 pm -> subsequent to meeting: introduction into lab
Summary 2012-05-12
Attendees: Basia, Stefan, Kerstin, Chris, Sascha, Tobias, Mario, Kathi, Kevin, Rico, Tom, Maria
1. pictuuuuuuures, group and single pictures were taken!
2. antibody
- response from Serge Muyldermans, possible antibody from Uni Brüssel
- no further news
3. presentation Biobrick assembly standard part 1: Basia
- RFC10
- RFC20
- RFC23 (Biofusion, Silver Lab)
part 2: Sascha
- RFC25
- RFC24
- RFC21 (Berkeley BBb format)
- RFC12 (Tom Knight BB2)
- Gibson Assembly
4. theoretical safety introduction
- lab manual with operating instructions, phone numbers, equipment manuals, order directions can be found under Protokolle
- instructions for virus group (15-05)
- A convinient date for everyone? Tuesday, 22-05 (6 pm) or Friday 25-05?
No meeting on Friday!!!
Next meeting: Tuesday 22-05, 6 pm
Summary 2012-05-22
Attendees: Basia, Stefan, Chris, Sascha, Kathi, Rico, Tom, Maria Protocol: Kathi Moderation: Basia 1. AID
- AID is delivered in distribution kit, will not be sequenced by us
- Chris talks again about the pTUNE- vector (see presentation)
- AID das fusion protein with fluorescence reporter for localization
2. Ordering
- can be organized and documented over wiki
- chemicals, reagents and consumable materials can be taken from the working group if not too much --> PLEASE do not contaminate when using
- a vacuum apparatus and a pipette boy is needed for cell culture
- Each team should present the partial group-project plan
3. start of lab work
- next week culturing cells will start, people with know-how should be assigned to cell culture group
- collection of vector and oligo material via Geneious, lab computer has Geneious account
- Geneious-version 5.1.7
Next meeting: Friday, 25-05, 8:15 am
Summary 2012-06-01
Attendees: Tobi, Laura, Sascha, Xenia, Basia, Kevin, Rico, Tom S., Tareck, Mario, Chris Protocol: Chris Moderation: Stefan 1. Organization
- ordering:
Ordering numbers and costumer ID can be found in the lab manual, all orders have to be documented on the wiki page The delivery note has to be collected and stored!!!!!
- iGEM-Distribution-Kit arrived, stored at -20°C in freezer
-> DNA should be tested by test digestion
- program Geneiuos has to be started from device C:\Programme... (version on desktop does not work)
- a list of needed plasmids should be generated
2. sponsoring
- application for financial help from the faculty was approved! We will possible receive the whole sum
- today: meeting with Sigma Aldrich rep -> list with needed materials
3.actuall progress report
- AID:
- next week: theoretical cloning -> expression vector for Biobricks?
- there already exist several eukaryotic expression vector in the lab
- AID construct: NES knock out & additional NLS, literature research has to be done to get further information on fusion proteins that make the AID more specific?
- virus team:
- meeting with Sven
- it has to be clarified if virus can infect cells
- plasmids construction should be planned
- antibody
- MTA for antibody should be signed
- problem with antigen: RNA-Polymerase, cannot be declared as therapeutically target
- possible solution: single chain that already exists can be used (lab stock)
- problem: already high affine binding (nM), should be mutated before to apply for affinity maturation via AID
- Kristina should order it from Freiburg
- suggestion: scFv coupled to Fc domain, good binding and good expression guaranteed
- still open requests for scFvs: Apergillus... advantage: affinity can be optimized
4. LMU München-Kongress Synthetische Biologie
- journey with overnight stay
- Mario?
5. BMBF Kongress in Berlin 28-06-2012
- people for representation (Tom, Kevin, Mario, Chris ), meeting the other iGEM teams
- a new poster has to be designed
Next meeting: Friday, 08-06, 8:15 am
Summary, 2012-06-08
Attendees: Maria, Rico, Tobi, Sascha, Xenia, Basia, Tareck, Mario, Chris, Kerstin
Protocol: Rico
Moderation: Tarek
1. virus group
- problem with primer design was discussed
2. orders for Eppendorf and Sigma Aldrich
- discount-list have to be completed on the wiki page
3. Congress BMBF
- Participants: Tom S., Tarek und someone from virus group
4. antibody
- further possibilities can be discussed:
- anti-EGFR (advantage: possible selection system for mutagenesis by AID can be tested)
- antibody against CRF2 (problem: sequence cannot be published)
- antibody against cholera toxin (problem: binding of pentameter )
5. German website
- first version is online
Next meeting: Friday, 15-06, 8:15 am
Summary 2012-06-12
Attendees: Mario, Laura, Chris, Basia, Sascha, Tareck, Tom S., Tom, Rico
Protocol: Xenia
Moderation: Maria
1. Sponsoring:
Application for StudiumPlus ready, no reply so far; document will be delivered on Monday (Maria)
Greenpaace will be contacted in Potsdam (Maria) and Berlin (Laura)
2. progress report
- Virus team
- primer construct leads to hair pins, should not cause any trouple due to high temperature
- second primer should have the same Tm and annealing temperature
- request for plasmid with CMV promoter in iGEM distribution kit (Sven?) – also necessary for AID group
- Xba1 restriction side has to be prolonged by 2 aa to guarantee the enzyme activity
- PolyA sequence present?
- primer construct leads to hair pins, should not cause any trouple due to high temperature
- AID:
- AID will be completed by NES (Nuclear exported Sequence)
- CMV (see virus team)
- AID will be completed by NES (Nuclear exported Sequence)
- Antibody:
- search for switchable system: cre-lox-system?
- idea for affinity measurement?
- search for switchable system: cre-lox-system?
- scFv anti-EGFR
LAB WORK:
CHO cell were taken into culture
3. prize and offer:
- prize list and chemicals were uploaded on the wiki page
- 25% discount for all enzymes of Thermo Fischer scientific
- Invitrogen GeneArt: 25 Cent pro base pair
4. BMBF
Poster for BMBF should be finished till 18-06!
Responsible for poster are: Tom S. (AID), Tarek (AK), Laura (Virus)
- Tom S. will design animation, Laura will write team info, Rico will write introduction
- People who will present the poster: Laura, Chris, Maria, Tarek, Mario, Xenia
- What should be presented on the poster?
- introduction (AID)
- explanation antibody, what was done so far?
- possible statistics for market potential of antibodies
5. time table
- end of September everything should be finished!!!!
- every group designs time table, publishing on internal wiki
- what do we have? What needs to be done and in which week?)
6. Phage Display
- expression vector can be taken from last year’s project
- EGFR domain will be produced by virus group
Safety assessment: Sascha, Mario, Chris
Next meeting: Friday, 22-06 8:15 am
Summary 2012-06-22
Attendees: Kathi, Stefan, Basia, Sascha, Kerstin, Tobias, Laura, Chris, Kevin, Tom S., Tom O., Xenia, Maria
Protocol: Maria
Moderation: Kevin
1. AID
- primer for Wt are designed and ordered
- AID from Biobrick Kit will have to be transformed (AID in mini backbone)
- vector psBIC3 with CMV promoter and RBS as glycerol stock
- quest for expression vector with restriction site between CMV and Poly A
Biobrick: assembly standard 25 a) CMV promoter + AID b) CMV promoter + kozak sequence + NLS + AID without NES time table for groups online
- verification of AID Biobrick
- competent cells have to be made (Monday)
- Basia will culture 50ml of ‘’E.coli’’
2. Antibody Antibody construct with: Signal peptide + scFv + Fc domain + LoxP site + transmembrane domain + reporter gene + LoxP site with Intron/Exon structure 3. Virus
- primer adjustment, ordering on Friday (22-06)
4. official beginning of lab work on Monday
- multistep-pipette is functional and can be used
- buffer for restriction enzymes received
> please complete table UP12_Materialllager
Important: using the labjournal and protocols for official presentation!
5. BMBF
- for all attendees: meeting for poster presentation and discussion about concept on Monday) am
Important: information about possible cooperation with other iGEM teams 6. Ethics
- Greenpeace and Nabu can be contacted
- further possible discussion partners: PETA?
- discussion about Synthetic Biology, gene manipulated organisms, animal testing for research and medicine
Next meeting for BMBF congress: Monday at 9 am
Next meeting in general: Friday 29-06 8:15 am
Summary 2012-06-29
Attendees: Tom O., Tom S., Kevin, Rico, Xenia, Kathi, Chris, Sascha, Tarek, Mario, Stefan, Tobi-as, Kerstin
Protocol: Kerstin
Moderation: Kevin
- competent cells in freezer (-80°C) and can be used
Report of the BMBF meeting
- poster of iGEM team uploaded on wiki
- possible cooperation with iGEM team Freiburg (DNA binding domain)
- suggestions for German wide ethic project: "Tag der Synthetischen Biologie" at the 25th August --> each team should organize something in their own home city
- coordination of day via Facebook
- free licenses for Geneious as sponsoring?
- antibody group presents construct and Biobricks
- planned meeting with genetic working group to talk about intron/exon sequence
Measuring the affinity by QCM? Important during the next weeks:
- progress report of each project; completing wiki information; ordering new lab material and con-sumables, should be ordered today (29-06) by Mario, Stefan, Sascha
- Next week Monday to Wednesday S1 inspection of whole Haus 25
Next meeting: Friday 06-07 8:15 am
Summary 2012-07-13
Attendees: Tom S., Rico, Tom O., Mario, Basia, Chris, Sascha, Xenia, Maria, Stefan, Laura
Protocol: Laura
Moderation: Sascha
1. poster
- Poster has to be revised, AID and antibody construct have to be added
2. abstract
- Should be finished and uploaded until the 15-07
3. expenses and lab inventory
- Sascha and Mario will actualize the table
- Waste and used items for autoclave were collected under the vent
- Tom S. and Rico will actualize list for lab material
4. time table of each group
- completing list of work steps and time table of each member
- phage display?!
5. Human Practice
- Akademie der Wissenschaften: possible cooperation, disadvantage: 150€ for 1 room and
- advertisement should be organized by ourselves
- new suggestion: presentation in schools or universities, Akademie der Wissenschaften will be contacted again
- asking for support in schools
6. workshop on 31-07.
- München poster should be used: Chris, Tom S., Laura
- Questionnaire can be spread along the attendees, collecting selection of questions
Next meeting: Friday 20-07 1:00 am
Summary 2012-07-20
Attendees: Tom S., Rico, Mario, Basia, Chris, Sascha, Xenia, Maria, Stefan, Laura, Kristian, Tarek, Kerstin, Tobi
Protocol: Basia
Moderation: Kathi
1. virus group progress report
- PCR annealing product with a size of 3000bp
- after digestion only 2000bp remained, additional restriction site?
- PCR product will have to be sequenced
2. EGFR Domain 3
- amplification: Ensemble-DB, Addgene.com, Origene.com, ?Qube.de -> as cDNA)
- testing for intron/exon structure
- 100-500 € with addgene
3. modeling
- Tobi presents modeling results
4. Cooperation with Freiburg iGEM team (Tal domain)
- we need a recognition sequence for Tal domain in our antibody construct
5. Questionnaire
- new questions should be formulated (German version)
6. iGEM - Tag der Synthetische Biologie 25-08
- Which steps should be organized? Where: schools- Potsdam city
- possibility of radio interview
7. ice cream :)
8. poster was printed today!
9. antibody construct is ready!
Next meeting: Friday 27-07 9:00 am
