Structure and Project Proposal:

• will be written in the Wiki
• catchy pictures have to be placed into the part Motivation / State of the Art
• Abstract has to be added

- hanger gold or NRPS? --> probably start with NRPS and outline gold-project as application

- depends on how many other teams choose gold-project

- gold into the project-title?

• outlook/final statement has to be added
• postpone costs, work on timeschedule
• methods have to be described more precisely --> look for experimental protocols
• what has to be formulated in the PP was written down in note form
• deadline for motivation part: Wednesday, April 17th evening
• general deadline: Monday, April 22nd evening
• deadline schedule, material + costs, final part + abstract: Wednesday, April 24th evening

Cloningmethods

• ask advisors for additional introduction to cloningmethods --> Nikos (Wednesday)

Laborplan Pre-project

• prepare ACM (1,5L + 0,5L (mit Agar)) Phytogel(NOT with Agar)
• reactivation solution (without agar)
• 0,5% agarose solution with ACM (maybe water) with 10mM AuCl3
• purification
  • HP 20 resin-beads: the Delftibactin attaches to the resin
  • Methanol dissolves the Delftibactin, afterwards dry (rotary vacuum)
  • resuspend it (in 2ml)

Motivation for Proposal (discussed with Tim & Tinka)

• central idea: NRPS (gold is the application)
• Weighing not clear
• start with gold
• Structure: Application = Framework
• Main part: relevance of NRPS
• generally more elaborate (not too much details)
• more numbers (tons of gld recycled)
• more structure
• why this pathway for gold precipitation and not another one
• don't use might or may
• softwareproject: how important is it for the gold?? why is it important??? judgement
• Trends of IGEM: no NRPS so far, thus new project for IGEM -> helps students and scientist in finding trends, etc...

3. Podiums discussion 27.6.13

• 18.00 talk about synthetic biology
• 18.30 discussion
• church, politics, scientists, NGOs(?) - 5 people
• Prof. Tanner
• first of all: make a concept with our goal
• catering afterwards (sponsoring)

Cloning

• BACs
• amplify genes via PCR (Problem, same Codons for E.coli and D.acidovorans)
• maybe not optimal codons
• Synthesis is not possible
• Biobrickstandard: separation in 1 Kb sequenzes
• first plasmids with one protein each
• then plasmids with multiple proteins
• and another try with whole pathway (BAC)
• now we need to find experts and ask them for help

Cloning Techniques/Strategies (Dominik)

• BBa (BioBrick Standard)
• Golden Gate
• Gibson Assambly

Discussion on Experimental Procedures

• Transformations
• Cosmids
• BACs

NRPS Library - with Discussion

• main ideas
• swaping of domains done already
• Q: Biology behind the System? (short presentation by Ilia on monday?)

Agenda for next meeting (29.04.2013)

• presentation of NRPS (library) (Ilia)
• presentation about methodology (BAC, PAC, Cosmide, Gibson Assambly (Fanny, Flo, Hanna, Joshua, Nicos)
• schedule (Hanna, Julia) ?

Ressource Collection

• Vector Design
  • Vector NTI
  • Geneious

• Gibson Assembly
  • NEB Discripton & Kit
  • Reference:

1. Gibson, D.G. et al. (2009) Nature Methods, 343–345.
2. Gibson, D.G. et al. (2010) Nature Methods, 901–903.

• Reserach on NRPS
  • AG Marahiel, Marburg --> Niels
  • AG Cryle, MPI Heidelberg --> Konrad

further planning

• Sat. 4. May: primer Design meeting ("Giant Inserts in E. coli")

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

Breakfast

• socializing =)

Catching Up on Lab Work (Hanna)

• news on the lab work
• present protocol

Gibson Cloning Primer Design (Nils)

presentation:

• Backbone pSB1C3, lacI, RBS, mRFP1, Cm-R, pMB! ori
• finding data for introduction of RBS-DelE (PacI and KpnI)
  • design of four primers
    • 2 for backbone
    • 2 for insert
  • go to parts registry, search pSB1C3
    • shows sequence and features --> get selected sequence
  • look at sequence in Serial Cloner
    • graphic map should show sequence and particular RE sites (bold names are unique sites, italic names are not existing sites)
    • look for cutting site that is not present in backbone nor insert/cassette
  • find cassette BBa_503495 sequence in parts registry (not standardized, not best example)
  • add cassette sequence to backbone sequence
    • show in graphic map
  • get DelE sequence (has a lot of PstI cutting sites, but EcoRI only a single site --> can be mutated)
  • Primer for adding RBS and cuttingssites between backbone and insert
  • If primer gets too large one can make two PCR steps
  • methyl specific cutting enzymes can digest backbone to separate the PCR amplified
  • Primer nomenclatur: Kürzel_Nummer:Protein_CuttingSite_fw/rev
  • Buffer of Restriction enzymes should be compatible
  • Forward primer protein: Overhang - cutting site e.g. PacI - RBS - 6bp (can be used to enhance specifity when taken from gDNA) - atg-Start of Protein - additional 22-27 bp for specificity (or smaller for big primers) - last one should be G/C, no repetitive G/C if possible
  • Reverse primer protein: Overhang - cutting site e.g. KpnI (reverse complementary in case of non palindromic enzymes) - reverse complementary end of protein
  • Primers should be same melting temperature / length
  • Watch out for first and second melting temperature and other reactions run in parallel!
  • Forward primer backbone: Overhang - cutting site e.g. KpnI - backbone specific sequence starting with RBS
  • Reverse primer backbone: Overhang - cutting site e.g. PacI - reverse complementary sequence of backbone until RBS
• Fold DNA on mfold:
  • 3' end should be accessible
  • 5' end can be varied (overhang)
  • wobble base pairs can be replaced
  • change length of primer to reduce folding
  • target would be a folding free energy > -2
• Run in silico PCR in serial cloner
  • target melting temperature between 60 and 70°C (if possible - not a critical parameter)
  • if you make mutagenesis the sequence is not complementary anymore!
  • Evaluate PCR chacks for complementary parts and gives product length
• Annotation
  • Features of sequence in serial cloner
  • in gene bank files

Ribosomal binding sites (Konrad)

• Current Plan:
  • add medium RBS for every part
• RBS in Del Cluster
  • RBS from Data base in file (Mail Konrad)
  • We can see the direction of transcription according to the primers

Use of J5 for Gibson assembly (Nikos)

• Parts in device editor all at once
  • Plasmid backbone
  • RBS
  • Several parts for Insert - every mutation is a single bp part
• Define how tot obtain the part
  • most of the PCR amplified
  • other option would be digestion
  • RBS would be embedded in a primer
  • Choose none for very small parts since it will choose lowest cost strategy
• Controls
  • Choose parameters
  • Choose Assembly meths
• Result is Vector Editor
  • Can show restriction sites
  • Primer sequences in csv file
  • Gives you cloning strategy

Agenda for next meeting (13.05.2013 18:00)

• One point per sub group
  • NRPS
  • Software
  • Del
  • Indi
• Define agenda for 15.5.

Group picture

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)