Team:Potsdam Bioware/Lab/Group Meetings

Contents 1 Group Meetings 2 Summary 2012-03-12 3 Summary 2012-04-04 4 Summary 2012-04-20 5 Summary 2012-04-12 6 Summary 2012-05-04 7 Summary 2012-05-11 8 Summary 2012-05-12 9 Summary 2012-05-22 10 Summary 2012-06-01 11 Summary, 2012-06-08 12 Summary 2012-06-12 13 Summary 2012-06-22 14 Summary 2012-06-29 15 Summary 2012-07-13 16 Summary 2012-07-20 17 Summary 2012-08-03 18 Summary 2012-08-06 19 Summary 2012-08-10 20 Summary 2012-08-14 21 Summary 2012-08-17 22 Summary 2012-08-21 23 Summary 2012-08-24 24 Summary 2012-08-28 25 Summary 2012-08-31 26 Summary 2012-09-04 27 Summary 07.09.2012 28 summary 2012-09-11 29 Summary 2012-09-04 30 Summary 2012-09-07

Group Meetings

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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?
    
• AID:
  • AID will be completed by NES (Nuclear exported Sequence)
    
  • CMV (see virus team)
• Antibody:
  • search for switchable system: cre-lox-system?
    
  • idea for affinity measurement?
    
• 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

Summary 2012-08-03

Attendees: Tom S., Rico, Chris, Sascha, Maria, Kristian, Tobi, Kerstin
Protocol: Kerstin
Moderation: Kristian
Discussion about team T-shirts Color? Logo will be presented in which size? Does everyone agree with the logo design?
science meets media Idea: creating website for human practice part of every iGEM team in the fashion of part registry
idea: radio interview for Tag der Synthetischen Biologie idea: certificate form ZEBIT/BFR?
- a new entry was created for the AID, should the entry be extended by a text about iGEM Pots-dam?
foto shooting and bbqing on Monday 06-08
who will participate in the Amsterdam jamboree? booking flights, hotel and so on.
antibody construct big construct: scFv will have to be displaced by anti-GFP nanobody small construct: almost ready, should be ligated into pcDNA5FRT vector for stable transfection
idea: IFP (infrared fluorescing Protein) with AID for measuring the mutation rate
Problems and limitations of FACS facility have to be assessed Alternative: Deutsches Rheuma Forschungszentrum with FACS core facility

Next meeting: Monday after foto shoot

Summary 2012-08-06

Attendees: Tom S., Rico, Chris, Sascha, Maria, Kristian, Tobi
Protocol: Tom
Moderation: Rico

• group planning:

(antibody): 1st week: small construct (transient/stable transfection, integrated 6th week), 3rd week big construct (transient/stable integrated 8th week)
(AID): 1st week: finishing everything, 2nd week: localization of modified AID, 3rd week: muta-tion rate with color change, (4th week) collaboration with Freiburg
(Virus): 3rd week: EGFR+ sortase construct and virus+ sortase construct, 5th week: virus is ready

virus: 3 variations of selection system: thymidin deaminase, antibiotic resistance, color reaction (dye should be compatible with GFP and mCherry)
phage display: Arabinose promoter fused to non-modified AID with Amp resistance, Tim should be consulted (analogy to NEB protocol)

Next meeting: Friday at 10:00 am

Summary 2012-08-10

Attendees: Tom S., Rico, Chris, Stefan, Laura, Tobias
Protocol: Chris
Moderation: Tom S.

• progress report:

(Antibody): search for Cre recombinase (Maria), important: cross interaction of cre recombinase and Flp-recombinase?
Big antibody construct will be too late for stable transfection till jamboree, can be transfected transiently, FACS for expression testing
Small construct is read for transfection, sequencing results will arrive soon, will be used for testing the AIDs mutation potential
(AID): weaker promoter for collaboration project with Freiburg, sequence specificity can possibly get lost with a high expression rate, first tests if expression with weaker promoter is useful
Phage display planning:
Plan: Transformation of ‘‘E. coli‘‘ with AID and phage vector, (new competent cells are needed) Induction of AID with arabinose 1 h before transformation of cells with vector Pak 100 (witht scFV 425- anti EGFR), adding helper phages afterwards
(Virus): selection for virus infection with which fluorescence protein? --> CFP? Leads to no inter-ferences with GFP,YFP and mCherry disadvantage: weak fluorescence

• Modeling: Tobias calculated and analyzed several parameter
  

Model shows that there should be a definite difference in affinity of template antibody to mutated antibody so that selection system can work properly (too high affinity of our antibody?)

• task allocations
  • Kathi: hotel and flight details for Amsterdam
    

Hotel: 2 nights, cheap, close to Uni
Flight: favorite way of travelling to Amsterdam

• • Tom S., Rico, Chris: new standard = ligation free cloning as "best RFC", Idea: cloning with

thiooligos

• • Kerstin: watching judging criteria
  • new Software protocols in XML format have to be written for Labapp (new protocols from lab work have to be used)
• wiki homepage: help by Norman (ideas and concepts should be collected)
• T-shirt design: 1st: uni chrome golden, golden with blue, any other suggestions?
• Tag der Synthetischen Biologie 25-08: ideas:
  • 1st: experiment with DNA extraction from bananas in pedestrian area (problem: permission by Potsdam city?)
  • 2nd: radio interview
  • 3rd: schools (problem: 25-08 is on Saturday)
• seminar presentations in English: each group should present a regular progress report to inform about lab work

Next meeting Tuesday 14-08 4 am

Summary 2012-08-14

Attendees:: Tom S., Rico, Chris, Stefan, Tobias, Sascha, Xenia, Maria, Bascha, Kerstin
Protocol: Tobias
Moderation: Xenia

• working time: time is getting short and there is still a lot to do
• group planning: stable transfection is getting started, RFP and AID will be co-transfected, fluorescence measuring over 48 h, control without AID, separating cells and sequencing
  

Someone has to contact the DRFZ FACS core facility
(virus): virus with YFP on surface are produced

• software: editor for protocol example is available; Basia tries to adjust protocols for software
• T-shirt: Tom designed several logos, Basia is responsible for imprinting of shirts (logo will be golden) everyone has to vote for the logo until next Tuesday
• human practice: (interview with a politician) two politicians already answered; formulating questions for the interview, everyone who would like to participate has to register with Name and date of birth
  

(radio:) Sascha contacted radio channels: no reply so far

• Tag der Synthetischen Biologie: DNA extraction in Potsdam, idea was accepted and should be planned

Summary 2012-08-17

Attendees:: Tom S., Stefan, Tobias, Sascha, Kerstin, Basia, Kathi
Protocol: Maria
Moderation: Kristian

• T-shirts:

- Design and color was set with red and golden letters (it still has to be decided if front and back site in uniform look or with different color for sponsoring)
- for every member 2 T-shirts were ordered, sizes were estimated (Basia immediately ordered shirts)

• Accessories:

- previous idea: flip-book, Buttons...
Further ideas?

• regional radio station were contacted by Sascha, no answer up to now

• Tag der Synthetischen Biologie

- stand at Brandenburger Straße was accepted by city of Potsdam, an official document is still missing (Maria will contact town hall again on Monday)
- information can be seen and collected under UP12_25_08_12

• 12th – 14th September: “8. Workshop of Molecular Interactions”
  

(http://www.molecularinteractions.de/)
Who would like to participate???? maybe 2-3 people for presentation
- a new abstract and a new poster is needed!

• Biobricks

Virus Team:
- improved/modified virus construct of Freiburg with sortase-motive
- antigen with sortase-motive
- sortase
- ...
AID Team:
- WT AID
- Super-AID
- Biobricks from Phagemid Display?
- AID-Tal Domain from collaboration
- ...
Antibody:
- entire antibody construct
- nanobody with Fc region
- TEV-LoxP- Transmembrane-domain-mCherry
- Cre Recombinase optimized for mammalian cells
- ...

• New iGEM standard by Rico, Tom, Chris
  

• Progress report: from next Thursday on, every group will have to present progress of lab work in English
  

- AID group (Tom S.): presentation of phagemid display (planning the vector and work steps), Biobricks, work for collaboration with Freiburg (AID+TAL Domäne)


Next meeting: 21th August 4pm

Summary 2012-08-21

Attendees:: Tom S., Chris, Stefan, Tobias, Sascha, Xenia, Maria, Basia
Protocol: Xenia

Tobias presents

• actual lab progress virus
• modelling

Basia presents

• actual lab progress AID
• TAL-domain
• standard cloning:

-PLICing or SLICing? – advantage of SLICe: fast, several inserts can be cloned at the same time ; cheap; cells already existent in registry? (quick and dirty)

• T-shirts: logo will be altered; t-shirts will have to be ordered

Day of Synthetic Biology (25.08.12)

• will take place at the Brandenburgerstr. in Potsdam next to Starbucks
• Topic: DNA isolation from bananas

• abstract will have to be extended

- dead line: 07-09-2012

Next meeting: 24th August 12 pm

Summary 2012-08-24

Attendees:: Tom S., Rico, Chris, Stefan, Tobias, Sascha, Maria, Kerstin, Kristian
Protocol: Kerstin

Agenda:

• work in progress
• poster und planning for Saturday 25-08 (Tag der Synthetischen Biologie)
• homepage
• 07th September
• interview questions
• transfection
• further planning

Discussion about official wiki on Saturday 25-08 at 5.30 pm
Until the 07th September all members should have signed in with an official wiki iGEM account
Track declaration, biosafety , transaction of registration fee for Jamboree
Progress report of antibody group by Sascha
Additional information about cell culture from Stefan and Kerstin
Maria presents poster for Day of Synthetic Biology (for Saturday, 25-08) Planning of Saturday, table is needed, lab coats, camera and so on…
All in all 7 interviews with politicians, dates can be inspected on wiki, interview questions have to be corrected again
Topics: flight and hotel for Amsterdam

Next meeting: 2012-08-28 12 pm

Summary 2012-08-28

Attendees: Rico, Tom, Tobias, Basia, Kerstin, Maria; Stefan, Xenia, Sascha Protocol: Sascha Agenda: 1. 07/09/12 2. payment jamboree/attendees 3. phone app 4. distribution of talks/progress report 5. workshop of molecular interactions 6. interview with a politician

1. 07/09/12

• safety questions, abstract and track selection is needed

2. payment jamboree

• StudiumPlus is sponsoring 1000€ for the attendance fee
• attendance fee 220€ pP

3. phone app

• Basia keeps trying to write protocols

4. progress report Antibody-group

• scFv construct was transfected into CHO cells, showed fluorescence
• not clear, if antibody is localized into membrane
• co-transfection of scFv construct with AID into CHO cells

AID

• checking sequencing results of CMV-AID_EGFP-hgH
• AID-TAL construct sent to team Freiburg
• phage display: cloning vector for BioBrick from Sven!

Virus

• PO1: YFP insert VP2/VP3
• PO5: VP2
• PO6: VP1
• helper phage: results looking good
• pSB1C: GOI and CFP+CMV

Modeling:

• 250h selection pressure on cells
• aim of model is to illustrate principle of project
• further parameters required to get a more precise analysis
• high selection pressure is needed for a good virus infection rate

5. molecular interactions

• new poster has to be designed
• poster presentation on the 12th of September in Berlin (Kerstin, Maria)

6. interview with a politician

• 8-9 interview partner showed interest

Summary 2012-08-31

Attendees: Basia, Stefan, Sascha, Kerstin, Chris, Xenia, Kathi, Rico, Tom
Presenter: Tom
Protocol: Maria
1.progress report:
Virus:
- pcBIC Plasmid is sequenced
- HEK cells are transfected with 4 plasmids for virus production (plasmid with YFP and CFP)
- Another GOI with “survival signal” should be designed!!
AID:
- All Biobricks (5 so far) are finished, got a number and are fully sequenced; just TAL domain is missing
- German names have to be translated
- Another Biobrick: Wt-AID-GFP = new standard can be used?
Phagemid Display:
- Difficulties with restriction enzyme digestion (was repeated in thermo cycler) and with purifica-tion/separation of single cut from double cut on gel
Antibody:
- Adjusting the transfection protocol is finished, transfection conditions are settled
- Clon 4 was transfected transient last week, pictures have to be taken with higher magnifica-tion
- But: fluorescence intensity was not high enough, repetition of experiment?
- Detection of antibody on surface with anti-FLAG-Tag antibody, secondary labeled antibody can be seen in microscope
- Start of long-time experiments with cmv-venus (48h over weekend)
- Cells are prepared for stable transfection
- GeneArt construct arrived!!!!
- Primers ordered for production of Biobricks
2. Episomal vector for longer transient transfection of antibody construct???
3. Measurement and data (needed!!!) for our project
- Mutations by AID: Sequencing of the antibody-fragment, search for sequence altered by AID
Idea: AID able to mutate specificity of antibody fragment for YFP to CFP ???
- Switch form on surface presented to secreted antibody fragment
- Selection system with virus
4. Virus
- Virus selection system needs to be able to distinguish between antibody/no-antibody on sur-face
- Possibilities of selection: death signal, survival signal (antibiotic resistance), fluorescence signal (CFP)

Signal	Surface	GOI
fluorescence signal	YFP	CFP
fluorescence signal	CFP	YFP
death signal	…	TK
death signal	…	CD
survival signal	…	antibiotic resistance signal which kills resting cells or use of selective media

- Find an antibiotic which is eligible for selection and orthogonal to Hygromycin
- Neomycin, Gentamycin????
5. Selection of CHO cells with antibody construct
- FACS of Potsdam antibody group still out of order
- Magnetic beats for selection
- FACS core facility at DRFZ
Important: phenol red free media has to be ordered for preparation
6. Assembly standard
- Biobrick standard vector, introduces compatibility
- Disadvantage: more expensive primers, ligase is still needed
- Advantage: much faster and vector doesn’t need to be amplified
7. Touch screen TV
- Collecting information
Idea: 4 smaller once instead of 1 big ones
8. Cre recombinase
– ordered from Addgene
9. Wiki/homepage
- Information/ ideas under UP12_ wiki-web-design

Important: suggestions for project name!!!!!

Next meeting: Tuesday, 04.09.12, 4 pm

Summary 2012-09-04

Attendees: Basia,Tom, Xenia, Tobi; Chris, Kathi, Maria, Stefan
Moderation: Tom
Protokoll: Kathi
Agenda :

• progress report
• abstract
• homepage
• FACS
• touch screen for poster presentation
• pictures

1. homepage

• Rico will provide HTML program
• labjournal for AID group can be uploaded on official wiki
• plug in for open office wiki?
• pull-down menu for wiki: max. 6 pull-downs
• content

- main: project description, data, summary 1, summary 2, summary 3, modeling - team - ethics/ human practice - safety - biobricks - data page - sponsoring -attribution -labjournal/protocols/ meetings - software 2. abstract

• will phage display be added to abstract?
• Maria will upload abstract, safety questions and tracks; text has to be controlled yet again

3. magnetic beads

• beads have to be coupled with CFP and YFP -> antibody junior group?

4. FACS

• pdf document for application for core facility at DRFZ (Maria sends mail)
• appointment can be made short dated
• people taking care of cell culture have to get involved

5. progress report

• Xenia presents current status of virus project (plasmids, future work…)
• gelfiltration
• virus has to be checked for fluorescence under the microscope
• positive selection with antibiotic resistance (which resistance gene is suitable?)

6. TV screen

• Basia has connections: 46 inches approx.. 1000€ + 250€ with screen, full HD, multi touch and laptop; cons: insurance just on the way to Amsterdam and back
• one touch screen approx.. 500€

Summary 07.09.2012

Attendees: Stefan, Kerstin, Maria, Basia, Kristian, Xenia, Tobi, Kathi, Sascha, Tom, Rico
Moderation: Tobi
Protokoll: Kathi
Agenda :

• progress report
• homepage
• FACS
• ELISA
• title/project name
• interview
  

1. title/project name:

• antibody evolution kit (system?)
• antibody maturation kit
• antibody generation kit
• new solution for antibody evolution
• best solution for antibody evolution

2. progress report

• AID: the phage display works
• VIRUS three out of four virus types are ready; last one is following next week; Primers for antibiotic resistance were ordered.... first virus infection was probably successful. A yellow shadow can be distinguished around the cells and after quenching the yellow glow disappears
• AK: stable transfection and Biobricks for single chain and nanobody construct in progress
• MODELING: Tobi presents new results for stochastic model – movie about correlation of initial amount of c with normal and und virus infected cells

3. homepage

• uploading of information onto official wiki via copy/paste - pictures will have to be added immediately with text to avoid server break down shortly before wiki freeze
• structure – generating as word document – will be adjusted later on

4. FACS

5. Magnetic beads: GFP is needed coupled to beads – Sascha is looking for other possibili-ties*Fluorescence BD homepage, checking out compatibility of fluorescence proteins with FACS facility

6. ELISA

• Sascha keeps searching for a labeled antibody against Flag-tag of scFv construct

7. interview with politicians (Tom, Rico, Sascha)

summary 2012-09-11

Attendees: Xenia, Tobias, Chris, Rico, Stefan, Basia, Kathi, Tom S., Kristian, Kerstin, Maria, Sascha
Protocol: Basia Tops:
1. Progress report
a) Phage display:
- the first round will be ready tomorrow
- 1 plate (double mutated with AID) had only 1 colony - maybe too strong mutation? -> let's try to reduce the arabinose concentration
- 3 others had many positive clones
b) AID group
- news from sequencing: wtAID mutates stronger than the mod. AID
- other probes from different times will be sent for sequencing tomorrow
- split the cells earlier?
c) Virus
- Things from Sven are there - available (missing plasmids and primers)
- Transfection tomorrow
- Do we do magnetic beads? Do we stay with Virus and FACS only as a selection system?
d) Antibody
- we need the cells with stably transfected antibody - Kerstin will do that until Friday but the cells won't be happy!
- Basia and Chris will express YFP and CFP for magnetic beads selection system
e) Modeling
- Comparison between deterministic and stochastic model: stochastic model is better
- we should apply the law of mass action
2. Biobricks - who writes what?
- Write an email to iGEM headquarters: is genebank format available? what features should it have?
- everyone should write the texts but only 2 or three people should put it on the parts registry - see the BioBrick page and self-assign
3. Internetseite
4. Poster
5. FACS
- Maria and Stefan will call the DRFZ.

Summary 2012-09-04

Attendees: Basia, Tom, Xenia, Tobi; Chris, Kathi, Maria, Stefan
Moderation: Tom
Protokoll: Kathi
Agenda :

• progress report
• abstract
• homepage
• FACS
• touch screen for poster presentation
• pictures

1. homepage

• Rico will provide HTML program
• labjournal for AID group can be uploaded on official wiki
• plug in for open office wiki?
• pull-down menu for wiki: max. 6 pull-downs
• content

- main: project description, data, summary 1, summary 2, summary 3, modeling - team - ethics/ human practice - safety - Biobricks - data page - sponsoring - attribution - labjournal/protocols/ meetings - software 2. abstract

• will phage display be added to abstract?
• Maria will upload abstract, safety questions and tracks; text has to be controlled yet again

3. magnetic beads

• beads have to be coupled with CFP and YFP -> antibody junior group?

4. FACS

• pdf document for application for core facility at DRFZ (Maria sends mail)
• appointment can be made short dated
• people taking care of cell culture have to get involved

5. progress report

• Xenia presents current status of virus project (plasmids, future work…)
• gelfiltration
• virus has to be checked for fluorescence under the microscope
• positive selection with antibiotic resistance (which resistance gene is suitable?)

5. TV screen

• Basia has connections: 46 inches, full HD, multi touch and laptop; cons: insurance just on the way to Amsterdam and back
• one touch screen approx.. 500€

Summary 2012-09-07

Attendees: Stefan, Kerstin, Maria, Basia, Kristian, Xenia, Tobi, Kathi, Sascha, Tom, Rico
Moderation: Tobi
Protokoll: Kathi
Agenda :

• progress report
• homepage
• FACS
• ELISA
• title/project name
• interview
  

1. title/project name:

• antibody evolution kit (system?)
• antibody maturation kit
• antibody generation kit
• new solution for antibody evolution
• best solution for antibody evolution

2. progress report

• AID: the phage display works
• VIRUS three out of four virus types are ready; last one is following next week; Primers for antibiotic resistance were ordered.... first virus infection was probably successful. A yellow shadow can be distinguished around the cells and after quenching the yellow glow disappears
• AK: stable transfection and Biobricks for single chain and nanobody construct in progress
• MODELING: Tobi presents new results for stochastic model – movie about correlation of initial amount of c with normal and und virus infected cells

3. homepage

• uploading of information onto official wiki via copy/paste - pictures will have to be added immediately with text to avoid server break down shortly before wiki freeze
• structure – generating as word document – will be adjusted later on

4. FACS 5. Magnetic beads: GFP is needed coupled to beads – Sascha is looking for other possibilities

• Fluoreszenz BD homepage, checking out compatibility of fluorescence proteins with FACS facility

6. ELISA

• Sascha keeps searching for a labeled antibody against Flag-tag of scFv construct

7. interview with politicians (Tom, Rico, Sascha)