Team:Potsdam Bioware/Lab/Group Meetings

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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

Team:Potsdam Bioware/Lab/Group Meetings

From 2012.igem.org

Contents

Group Meetings

Summary 2012-03-12

Summary 2012-04-04