Team:Heidelberg/Templates/M-15-05-13

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Contents

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