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From 2013.igem.org

Contents 1 Gold 1.1 recapitulation of NRPS domains and modularity 1.2 what has been done 1.3 NRPS biobrick library 2 Coloured NRP: indigoidine (Ralf) 3 Cosmide (Hanna) 4 Phagemids & PACs (Flo) 4.1 Phagemids 4.2 PACs 5 BACs (Fanny) 5.1 Recombineering 6 Gibson cloning (Nikos)

Gold

• we were able to reproduce the paper --> images with gold
• delftibactin purification didn't work by now --> try again

recapitulation of NRPS domains and modularity

• domains that are essential: C: Condesantion, A: Adenylation, T: Thiolation

what has been done

• simplification of synthases (smaller linkers) -> no negative effects
• replacement of alanin to serene by changing domain
• module extention
• short de-novo NRPs

NRPS biobrick library

• produce a library with different combinable NRPS-subunits
• start with producing staphcillin (has already been done --> realistic to achieve)

Coloured NRP: indigoidine (Ralf)

• we could prove the modularity of our NRPS-library by producing indigoidine
• is a blue dye
• synthesized from 2 gylcin molecules with a certain enzyme
• there are only a few cutting sites in the required genes

Cosmide (Hanna)

• insert size 50kbp
• has got cos site --> in vitro packaging
• has got only very few cutting sites (BamH1)
• selection maker: ampicillin
• check of digestion with 0.8% agarose gel
• preparation with phenol/chloroform extraction

Phagemids & PACs (Flo)

Phagemids

• you can infect cells with phages or by electroporation
• 10-20 kbp
• high yield of DNA
• E. coli have too be competent for F-Phages
• DNA extraction via phages

PACs

• p1 artificial chromosomes
• hybrid system of plasmid and p1 phage replication
• insert size 70-150 kbp
• transformation using phages
• isolation with kit possible (QIAGEN)
• good transformation-rate but complex system
• you have to be very careful working with phages --> easy contamination

BACs (Fanny)

Recombineering

• well established system
• 150-350 kbp
• low copy numbers
• "lamda red recombination" using 3 viral genes: 5'-3' exonuclease, overhang binding protein, inhibitor of bacterial exonuclease
• use of cassette with pos and neg selection marker
• electroporation of gene of interest
• cloning: cassette is brought into the BAC by homologous recombination --> also several times possible
• there is a library of E. coli strains which already have the cassette

Gibson cloning (Nikos)

• iGEM biobrick standard will be hard for us to realize --> delH
• we will promote modularity with the NRPS library
• for the gold project we will likely use gibson cloning
• we will talk about primer design on saturday (4th May)
• start PCR as soon as possible