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From 2013.igem.org

Contents 1 State of the lab 1.1 backbones 1.2 DelH 2 Further plan 2.1 methylmalonyl-CoA-strain 2.2 DelH 2.3 Indigoidine and NRPS library 2.4 Gibson assembly 3 Gibson approach, Indigoidine and NRPS library 3.1 DelA-P Cloning strategy 3.2 Indigoidine 3.3 Washington pGA 3.4 standards... 3.5 conclusion 4 Software 4.1 iGEM pulse 4.2 NRPS software 5 Budget 6 Miscellaneous 7 Conclusion 8 Agenda for next meeting with Advisors (06.06.2013)

State of the lab

backbones

• psB4K5 for Del A-P is ready
• psB6A1 for DelH not
• psB1C3 for Indigoidine is ready

DelH

• two parts via SalI (8 kb and 10 kb) successfully amplified
• will be integrated into plasmid for electroporation in E. coli

Further plan

methylmalonyl-CoA-strain

• K207-3 is not available
• we get BAP1
• Lambda-red plasmid
• FLP plasmid
• Template plasmid

DelH

• Expression in E. coli end of may

Indigoidine and NRPS library

• problems concerning RFC10
• Gibson Info Freiburg required
• Washington standard: contact Washington team! (Fanny?)

Gibson assembly

• maybe method of choice
• are there protocols or do we have to delelop standard protocols
• small fragments could be a problem (PCP-domain)
• develop Gibson RFC standard for NRPS library?

Gibson approach, Indigoidine and NRPS library

• problems with RFC10 restriction sites and gene size

DelA-P Cloning strategy

• leave out not necessary genes like DelC
• five cluster fragments
• use Gibson cloning
  • Dominik: mid of next week we will get further information
  • avoid too big PCR-constructs; 10 kb has been very difficult to amplify

Indigoidine

• try plasmid, reproduction of paper
• seperate domains (A, T, TE) and fuse again to proove modularity
• maybe exchange domains with domains from other pathways
• Müller Plasmid -> new problem of 11 restriction sites
• How do we want to assemble NRPS modules in our project?
• maybe Berkeley RFC21 standard because it works very good for our gene clusters
  • compatible with linkers
  • allows fusion protein
  • not accepted by iGEM registry
  • BglIII cannot be heat-inactivated
  • benign scar (GS)

Washington pGA

• Gibson Assembly Toolkit
• based on RFC21 backbone

standards...

• mix and match domains not using biobrick standard but another strategy
• it is too much work to get our parts conform to RFC10 and the NRPS-pathways are not realy compatible with RFC10 at all
• after first experiments consider codon optimized synthesis for part submission
• other NRPS not standardised, but propose a new standard
• few characterized parts are of more value than a hundret useless ones

conclusion

• why not omit RFC and start with Gibson at the first point
• get expertise in Gibson cloning (read papers)
• Hanna and Julia (TA) should try Gibson assembly together

Software

iGEM pulse

• use scrapy (python) to get data from last years wikis -> MySQL (first subgroup)
• develop parameters for iGEM oulse webpage (second subgroup)
• First Deadline: May 24
  • detailed plan on data mining and parameter design
• Second Deadline: End of June
  • valuable product

NRPS software

• NRPS library software starting after proof of cencept in Indigoidine
• predict required domains for input structure and propose cloning protocol

Budget

• possible sponsors
  • Klaus Tschira as main sponsor
  • talk to other sponsors for small fundings (connections...)
  • university funding, helmholtz, biotech,
• sponsorship benefits?
  • logos on Shirt, Website, presentation

Miscellaneous

• reservation of lufthansa flights for Boston!
  • group tickets are cheaper
  • greater flexibility
• book hotels/ guest houses in Lyon!
• marketing will be done by professionals
  • They will need the project description with loads of random pictures (domains, gold, electronic waste, cash, ...)
  • will be there at the next meeting

Conclusion

• start Indigoidine subproject with Gibson cloning (faster!)
• take care about hotels, flight (Sophie)
• project proposal, sponsor list (coordinated by Fanny)
• creative input for marketing people

Agenda for next meeting with Advisors (06.06.2013)

• talk to marketing people
• progress in labwork
  • DelA-P
  • Indigoidine
  • DelH
• travel agency
• project proposal, sponsor list