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

From 2013.igem.org

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)