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

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Contents

NRPS subgroup (Julia, Philipp)

  • Possible cloning standards
    • Handing in biobricks only in pSB1C3 (RFC10 standard)
    • RFC25 (scar 6bp) standard because RFC10 scar (7 bp) causes frameshift
  • Parts to be cloned from Tyc
    • T domains either in front of E or in front of C --> cloning 1 per group
    • A and C as single domains and as couple in parallel
    • E domains and Thioesterase
  • Cutting sites
    • not in Tyc A/B/C SpeI, EcoRI, XbaI, NgoMIV
    • Tyc B NgOMIV in C domain, in T-C linker
    • Tyc C PStI in CIII domain, NotI in AIII domain

Indigoidine subgroup

  • Plasmid optimised for eucariotic cells
  • Impossible to clone it as RFC10
  • Use RFC21 instead -> compatible with Tyc?
  • first as whole part
  • Can be transformed wihtin in this week
  • primer design within this week

Cloning standard discussion

  • Gibson!!!
    • find good reason, concept for Gibson cloning
  • standard cloning techniques unfeasible
  • Further plan
    • Order all he plasmids and strains for permeable strain (BAP1 strain, red plasmid, FLP plasmid, template plasmid)
    • Indigoidine (Ralf) RFC 21 + check for other NRPS (Ilia) pathways
    • Modularity of Indigoidine RFC 21
    • in parallel check on Gibson assembly in Freiburg (Dominik)
    • get Backbones from Washington (Fanny)

Software subgroup (Konrad)

  • subgroups for data mining, data management, statistical analysis, front end
  • right now mainly building data management + concept for frontend and analysis
  • starting with NRPS software only makes sense as soon as NRPS gives any positive results
  • next one and a half weeks for being productive and then we decide for or against the project

Delftibactin subgroup

  • 8 and 10 kb successfully amplified
  • digest tomorrow
  • Ligating AraC promotor this week
  • If cloning in E.coli doesn't work we skip Del or search for different organism (next week earliest)
  • definitely no restrictiton based cloning -> depending on Gibson cloning technique

Miscellaneous

  • Send EMail to registry: How to submit parts? , Promote system open for new standards. (Fanny)
  • We should find a plan B for Delftibactin!
    • in vitro parts expression, ex vivo delftibactin synthesis?

Agenda for next meeting (15.05.2013)

  • State of the lab (Hanna)
  • Further plan (Fanny)
  • Gibson approach (Flo, Nikos)
  • Enzymatic cloning and dependencies of NRPS and indigoidine (Nikos)
  • Software project deadlinev + plan (Julia, Philipp)
  • Budgetplanung (Prof. Eils)