Team:Potsdam Bioware/Lab/Group Meetings

Group Meetings

Summary2012-03-12

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