NRPS – Delfibactin project
by Ilia
1. delfibactin:
- export from biocompartiments: unspecific outer membrane transporter (so delfibactin gets into medium)
- synthesis: pathway known, e.coli strain for production (maybe not clone DelC)
2. NRPS: introduction
- modular enzymes: 3 modular domains (together 110 kDa)
- steps: adenylation, acylation, condensation
3. software: input
- match amino acid to known A-domain substrates
- recognize easy modification (in amino acids) for which substrate you need it
- method: substructure based algorithm for searching and predicting substrate specifity
- it works in a R-package
- 10 amino acids are important for the substrate specifity
4. biobrick library
- there is no standard for the Biobricks of NRPS
GHB
by Flo and Nils
1. Jugend forscht: done a test (we do not understand how it works)
- not reached the supervisor yet
- she didn't win anything
- detect it via Spectrometry
2. 3 ideas for integrating the receptor
- artifiial membranes are not an option (you can't predict how the protein is located)
- in vitro expression of GPCRs (takes too long)
- Luciferase activity under directt ligand-dependent control of muscarinic acetylcholine receptor
=> measurments possible (very sensitive: represses glowing a little bit, a bit more... but never completely)
(integrate it is fast, but the mutagenesis takes a long time)
Artificial yeast-cells for sensing Insulin-level and producing/distribute the Insulin
by Flo
- presentation of the idea and past igem projects regarding this theme
- semipermeable biocapsules (with cells inside)
- the cells (yeast , bacillus subtilis, e.coli... ) for producing insulin if it is necessary
- there exists a famous lab for synthetic biology which works on insulin production
Software Project
by Julia, Konrad and Hanna
- parameters for statistical analysis and how they correlate (like teammembers, advisors, organsims, score regarding a scoring funtion)
- graphics: basically histograms
work for the next weeks
1. software project
- look on the focus of the team!
- better definition of the parameters
- estimate the cost of an IGEM project (there exists something for the nature paper... just looking at the methods etc...)
- already made something with these parameters
- clustering for example, not only directed
- software project should be finished before the lab phase: end of June/ July
2. Gold Project
- working on this project: Nils, Ilia, Julia, Ralf, Hanna
- find stuff we have to order and send Dominik an email => done!
- talk to companies (interested, is it done already and how, are there established assays)
- find succesful examples
3. general information
- we have to register and then apply for the team. LogIn
- send an email for getting the contact details (parameters which here already used)
Agenda for next meeting (29.04.2013 at 5 pm)
- results of pilot project (delfibactin in the lab)
- present a project plan (goals, time etc.)
- update on the software project. How to proceed etc.
Structure and Project Proposal:
- will be written in the Wiki
- catchy pictures have to be placed into the part Motivation / State of the Art
- Abstract has to be added
- - hanger gold or NRPS? --> probably start with NRPS and outline gold-project as application
- - depends on how many other teams choose gold-project
- - gold into the project-title?
- outlook/final statement has to be added
- postpone costs, work on timeschedule
- methods have to be described more precisely --> look for experimental protocols
- what has to be formulated in the PP was written down in note form
- deadline for motivation part: Wednesday, April 17th evening
- general deadline: Monday, April 22nd evening
- deadline schedule, material + costs, final part + abstract: Wednesday, April 24th evening
Cloningmethods
- ask advisors for additional introduction to cloningmethods --> Nikos (Wednesday)
Laborplan Pre-project
- prepare ACM (1,5L + 0,5L (mit Agar)) Phytogel(NOT with Agar)
- reactivation solution (without agar)
- 0,5% agarose solution with ACM (maybe water) with 10mM AuCl3
- purification
- HP 20 resin-beads: the Delftibactin attaches to the resin
- Methanol dissolves the Delftibactin, afterwards dry (rotary vacuum)
- resuspend it (in 2ml)
Motivation for Proposal (discussed with Tim & Tinka)
- central idea: NRPS (gold is the application)
- Weighing not clear
- start with gold
- Structure: Application = Framework
- Main part: relevance of NRPS
- generally more elaborate (not too much details)
- more numbers (tons of gld recycled)
- more structure
- why this pathway for gold precipitation and not another one
- don't use might or may
- softwareproject: how important is it for the gold?? why is it important??? judgement
- Trends of IGEM: no NRPS so far, thus new project for IGEM -> helps students and scientist in finding trends, etc...
3. Podiums discussion 27.6.13
- 18.00 talk about synthetic biology
- 18.30 discussion
- church, politics, scientists, NGOs(?) - 5 people
- Prof. Tanner
- first of all: make a concept with our goal
- catering afterwards (sponsoring)
Cloning
- BACs
- amplify genes via PCR (Problem, same Codons for E.coli and D.acidovorans)
- maybe not optimal codons
- Synthesis is not possible
- Biobrickstandard: separation in 1 Kb sequenzes
- first plasmids with one protein each
- then plasmids with multiple proteins
- and another try with whole pathway (BAC)
- now we need to find experts and ask them for help
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