Team:Heidelberg/Meetings

From 2013.igem.org

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Revision as of 03:24, 5 October 2013

Meetings.

We met regularly together with all our advisors and instructors to discuss the project strategy, to distribute tasks and to initilize additional social events.

NRPS – Delfibactin project

by Ilia

1. delfibactin:

  • export from biocompartiments: unspecific outer membrane transporter (so delfibactin gets into medium)
  • synthesis: pathway known, e.coli strain for production (maybe not clone DelC)

2. NRPS: introduction

  • modular enzymes: 3 modular domains (together 110 kDa)
  • steps: adenylation, acylation, condensation

3. software: input

  • match amino acid to known A-domain substrates
  • recognize easy modification (in amino acids) for which substrate you need it
  • method: substructure based algorithm for searching and predicting substrate specifity
  • it works in a R-package
  • 10 amino acids are important for the substrate specifity

4. biobrick library

  • there is no standard for the Biobricks of NRPS

GHB

by Flo and Nils

1. Jugend forscht: done a test (we do not understand how it works)

  • not reached the supervisor yet
  • she didn't win anything
  • detect it via Spectrometry

2. 3 ideas for integrating the receptor

  • artifiial membranes are not an option (you can't predict how the protein is located)
  • in vitro expression of GPCRs (takes too long)
  • Luciferase activity under directt ligand-dependent control of muscarinic acetylcholine receptor

=> measurments possible (very sensitive: represses glowing a little bit, a bit more... but never completely) (integrate it is fast, but the mutagenesis takes a long time)

Artificial yeast-cells for sensing Insulin-level and producing/distribute the Insulin

by Flo

  • presentation of the idea and past igem projects regarding this theme
  • semipermeable biocapsules (with cells inside)
  • the cells (yeast , bacillus subtilis, e.coli... ) for producing insulin if it is necessary
  • there exists a famous lab for synthetic biology which works on insulin production

Software Project

by Julia, Konrad and Hanna

  • parameters for statistical analysis and how they correlate (like teammembers, advisors, organsims, score regarding a scoring funtion)
  • graphics: basically histograms

work for the next weeks

1. software project

  • look on the focus of the team!
  • better definition of the parameters
  • estimate the cost of an IGEM project (there exists something for the nature paper... just looking at the methods etc...)
  • already made something with these parameters
  • clustering for example, not only directed
  • software project should be finished before the lab phase: end of June/ July

2. Gold Project

  • working on this project: Nils, Ilia, Julia, Ralf, Hanna
  • find stuff we have to order and send Dominik an email => done!
  • talk to companies (interested, is it done already and how, are there established assays)
  • find succesful examples

3. general information

  • we have to register and then apply for the team. LogIn
  • send an email for getting the contact details (parameters which here already used)

Agenda for next meeting (29.04.2013 at 5 pm)

  1. results of pilot project (delfibactin in the lab)
  2. present a project plan (goals, time etc.)
  3. update on the software project. How to proceed etc.

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

  • 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.

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

Cloning

  • DelH (review of the work done so far)
    • primers were designed and ordered
    • DelH gene first try with plamsid backbone: pSB6A1
      • AraC Promoter, RBs, lacZ, low ori pMB1, amp-R
      • PacI and KpnI are not in DelH gene, thus used for ligation
    • DelA to P into single plasmid with about 26kbp: pSB4K5
      • lacI Promoter, RBs, mRFP1, low ori pSC101, Kan-R
    • Hanna's work so far: Top 10 E.coli
      • pSB4K5 with Insert
      • pSB6A1
      • AraC
      • lacZ
      • pSB1C3 with insert 
  Are supposed to be joined by cloning to complete backbones by Monday
  • Indigoidine
    • similar to DelH
      • pSB1C3
      • high ori 0034, lacI promoter, mRFP1, Cm-R
  • cloning with +TypeII RE (recognize sequence and then cut at site one nucleotide next to recognition site)
    • division into three fragments

ask Dominik:

  • how exactly does this strategy work?
  • when is this an appropriate strategy to use?
  • Is "PCR Ligation" also a good Method (seperate PCRs for fragments; primers have "overlapping tail" takend from next fragment; ligation of fragments in one single PCR")?

Important! What about Freiburg? are they willing to help us with Gibson Cloning?

Approach of the Next Few Weeks

What should be bought: ---> Konrad's list

  • divison into smaller groups dealing with the main topics of our projects:
  1. Indigoidine (closed): Rald, Konrad, Nikos
  2. Del H lab protocol/methods (closed) : Hanna, Fanny, Sophie
  3. Del Rest (A to P) (closed) : Flo, Ilia, Nikos, Nils
  4. NRPS : Tania, Joschua, Ilia
  5. Software : Nils, Ilia, Konrad, Joschua, Hanna

Decisions Made

  • serial cloner should be our main software used for primer design etc.
  • Should use tool for oligo 2ndary structre prediction
    • http/mfold.rna.albany.edu
  • Should create common primer list; structure as in the following:
    • continous number, primer name, abbreviation (initials), sequence, details (if possible with a link to protocol used), creator, date, evaluation (e.g. worked, low yield ... )

Agenda for next meeting (13.05.2013 6 pm)

  • all groups meet up seperately and continue working
    • brief presentation on next meeting
  • next meeting (with Dominik)
    • progress report of individual groups and lab
    • prepping for upcoming big meeting
  • Next big meeting will be 15.5.2013

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

File:Protocol 09-05-2013.jpg
Breakfast!

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)

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

No english protocol available.

Power Cut

Power was down for 10 min in Labs --> Lab Materials should still be fine.

Labupdate

DelH

  • DelH fragments are ready for ligation
  • LacZ, AraC --> Cut again and purified
  • Backbone --> New Backbone is being amplified in bacteria taken from stock

Plan: Purify, Digest and PCR of backbone (could then be ligated with DelH on wednesday)

Indigoidine

  • Transformation of SVP and Bps in individual E.coli succeeded

Plan: Get BL23C3 and cotransform both Plasmids

General

  • Partsregistry kit shipping: At the moment some problems with customs
  • Lab materials will be ordered tomorrow

To do:

  • Document glycerol stocks etc.
  • Nikos: Ask Silvan and Jan if we can have script for getting PCR-protocols of PCR machine
  • Hanna: Doodle for date for arranging everything in the lab

Symposium BMBF

  • 27th of June
  • Human practice?
  • Poster has to be made --> don't put too much effort in it
  • Nils starts creating a poster

Who wants to go?: Hanna, Philipp, Nils, Fanny, Tania, maybe Konrad?

Software

Statistics

  • Script which gets data from databank to R is ready (uploaded on Github)
  • Next step: designing apps

Webdesign

  • rApache (Ilia) vs. Shiny (Nikos)

Cost analysis

  • Complete one is not feasible
  • Instead: Only analyse single methods (can be chosen by user, website returns protocols, costs, teams that have used the method)
  • Next step: Look for methods, which could be analysed

General

  • Which is our target group??
  • Connect iGEM pulse with our project (for example evaluate our team with it)

Sponsoring

  • Template mail for sponsoring is nearly ready
  • Template project proposal of Eils didn't arrive yet

No english protocol available.

Introduction Marketing

  • Gold versus NRPS-library
  • entirely new design but link to the last ones

Update labwork

  • backbones ready

software

  • plans for apps and analysis
  • cost analysis tool
  • front end

indigoidine

  • shuffle T-domains as proof of modularity

Sponsoring

  • brochure for sponsors
  • we need a group photo

next meetings

  • barbecue: june 10th
  • with advisors: june 12th
  • with supervisors: june 19th

sub-groups

how do we organize sub-group-meetings?

one group 100 % or two groups (1st group 70% and 2nd group 30%)
groups how many persons do we need? main person who works here?
IGEM42-software 1 Julia Julia, Philipp, Ilia, Anja, Nikos, Konrad, Hanna
NRPS-library-software 3 Ilia Ilia, Nils, Joshua, Nikos, Konrad, Julia
Indigoidine/Tyrocidine 4 Ralf Ralf, Ilia, Joshua, Flo, Nikos, Konrad, Sophie
Delftibactin 2 Hanna Flo, Sophie, Anja, Nils, Philipp, Hanna
Human Practice 1 Philipp Philipp, Fanny, Hanna
Wiki 1 Ralf & Flo Ralf, Flo

NRPS-library

    1. how can you synthetisize your wanted protein?
    2. which modules do you have to shuffle for this protein?
    3. evaluate the modules? which modules are better than the others? => pathway should be with modules of ONE organism.
=> prepare for wednesday a presentation of concept (Nils & Ilia)= Topic 1 (name) =
  • result1
  • result2

Agenda for next meeting (17.06.2013)

  • subgroup presentations
  • NRPS-library - present a concept
  • hotel and flights to Boston

Logo Presentation Lange und Pflanz

  • couple of proposals, which partially stick to the James Bond style and partially don't
  • the Team chose logo 2
  • there were additional comments to the logos
  • after a second vote logo 3 was chosen

Sponsoring

  • we have to write into the application what we want from the companies!

Slovenian Company

  • Prof. Eils met people of a Slovenian company, which focuses on transferring very big amounts of DNA (up to 200kb) from one kind of bacteria to another one

DelH Cloning

  • DelH colonies grown after 24 h (two big and one small colony)
  • No blue colonies
  • Anja will replace Fanny and Hannah in the lab until friday
  • Suggestion: check on blue colonies only with easy backbone ligation

Indigoidine

  • Probably received the right plasmids
  • results not as good as in paper (not as blue)
  • BAP1 + plasmid is stronger
  • Gibson cloning of activating gene and NRPS on one plasmid
  • Tomorrow miniprep on picked colonies
  • Suggestion: Check expression of ribosome binding site with mCherry-lac system

Genomic integration

  • Probably not enough DNA -> PCR with new primers
  • Colonies definitely negative
  • making fresh competent cells

Software

  • Several deadlines for different components
  • Deadline 8.7.
  • text mining should work
  • currently working on components and putting them together
  • next meeting on 1.7. 19h at Julia

Miscellaneous

  • NRPS estimation of computational feasibility
  • Project proposal - lots of feedback -> will be finished tomorrow evening
  • NRPS library should maybe get some more space in proposal -> Philipp until tomorrow
  • If we lack time in the lab we have to pe prepared properly including all primers.
  • We should have an internal meeting before tuesday to discuss about motivation, time we want to spend.

Agenda for next meeting (2.7.2013 19h)

  • Report Berlin (Philipp)
  • Lab update (all lab people)
  • iGEM42 (Julia)
  • Motivation & time schedule
  • Sponsoring update

Agenda for next meeting (3.7.2013 18h)

  • Lab update (all lab people)

iGem42 - input on Design:

  1. no greek
  2. round edges
  3. one uniform lettering
  4. design: where am I?
  5. dark blue, rest modern

=> 3 different designs until tuesday

lab update

  1. general
    • polymerase dosn't work as expected (Q5 in contrary works)
    • Phusion Flash from advisors works also
    • CyclerProblems: Gradienten-PCR doesn't work
  2. indigoidine
    • streptomyces strain arrived => monday culture the streptomyces
    • plasmid containing sc-indc is ordered
    • primer monday to Dominik and then order
  3. DelH
    • colonies after electroporesis had no integrated DelH
    • amplification of DelH fragments again didn't work - only with Phusion Flash (1b & 2)
    • Backbone is not amplified (pSB6A1-AraC-lacZ)
  4. Del-Rest
    • Primers are ordered
    • since wednesday: Del-rest amplify from the genome (Flo & Nils)

organization

  1. Google-Kalendar for all
  2. responsibility of ordering stuff: Sophie
  3. create Milestones & lab managment on the wiki: Sophie
  4. tyrocidine has to be started = parts for sending in (check-liste)
  5. wiki team (Ralf, Flo und Sophie) check the list for gold medal!

Agenda for next meeting(02.07.2013)

  1. Milestones - Groups make the Milestones for their group erstellen diese mit Gruppe
  2. timeschedule ?who, when?
  3. iGEM42
  4. Sponsoring update

  • new logos were presented to us by Kai
  • the majority and Prof. Eils of the team supported the 3. logo (which is more complex, represents modularity)
  • Kai continues to work on the logo (colors, animation)

iGEM42 & Website

  • Julia presented 3 proposals of possible websites
  • we should use one layout for all websites/wikis
  • Kai will send us his background from the logo-presentationns

Milestones

  • we went through all milestones with Prof. Eils

Thermocycler-problem

  • talk to the IPMB!
  • find a solution for next two weeks --> Tinka's lab (plan in advance and let her know!)
  • we will propably buy 1-2 new cyclers

Slovenian Company

  • talk to them for Del-transformation

Sponsoring

  • Steimbuch Stiftung (Software founding up to 10.000€) might be interesting
    • Prof. Eils: the timeline is pretty long
  • Claus Tschirra is most important
    • Fanny will send a corrected version of our project proposal to Prof. Eils

Human Practise

  • Stage discussion: on synthetic biology...
    • after European Jamboree
    • Prof. Eils: human practise is important! but we shouldn't overdo it
  • go into schools
  • science & arts --> bioquant is already in some cooperation

Cooperation with Freiburg(Hanna)

  • arrive on Monday(14.7.) want to show us Gibbson Assembly
  • accomodation: Philipp, Julia, Hanna/Ralf
  • Would go out for Dinner at Schmidt's, Hanna is going to send an Email
  • we should take away all the things in the lab indicating our actual project with Delftibactin

Lyon (Hanna, Philipp, Tania)

  • hotel: 7x 3 people in one room
  • train to Lyon is booked, need to book the ride back on 15.7. (Monday)

Human Practice

  • Konrad offered to take care of the soil samples to send to the UK (?)
    • want to get some info on Streptomycces from them.
  • Fanny and bts take care of the discussion round

Financing, Karl Steinbruch Stiftung, Software Project

  • Anja: we need three people's CVs that have worked in Bioinfomatics and have more than 120 ECTS
  • they want to know about where we spent the money
  • max ammount is app. 830 €/month
  • dead line is 31.7.

Lab Update

Delftibactin

  • Del H
    • problems with 1B bands
      • new amplification worked
      • were digested and purified today
      • are going to be ligated tomorrow

Dominiks suggestion:

  • divide ligation after 1 hour into two, one is kept at -20°C the other is let continue over night at 4°C
    • pool both together and electroporate this batch
  • gibbson primers arrived today; this will be the new approach, if ligation is unsuccessful
    • parallel gibbson does not make sense, to expensive

---> gel is running right now and will influence decision on further proceeding

  • Del Rest
    • 7-8 fragments of 32 are still missing
    • backbone has been amplified
    • Flo designed new primers
      • for DelOP

Indigoidine

  • pptase could be amplified, but not indigoidine synthetase
    • brainstorming
      • maybe the gene is not in this sub-species?
  • plamsid from UTAH arrived
  • want to write to other labs if they can send us the plasmid
  • there is a different Streptomyces (Stamm) that has this enzyme but need to finde the correct name
  • burocracy discussion.. MTA, Hauspost, Eils signature
  • primer strategy with BPSA

Tyrocidine

  • Stamm come from Marahije (?)
  • Glycerol stocks,colony PCRs
  • new strategy has be developed
  • ordering of primers by end of the week

Methyl-malonyl CoA

  • want genomic integration of substrate synthesis complex for DelF
  • got the insert from the USA
  • just need to amplify the insert with appropritate primers with some extra DNA
  • first tries didn't work
  • has several primers for accurate testing/screening
  • if integration worked only one resistance gene copy
  • gets fragment PLUS other bands of something else...
  • did electroporation and purification this week
    • some altered conditions
  • some good colonies
  • screening

NRPS

  • primary goals of the NRPS etc
  • NRPS are modulary
  • three domains, CA-T-optional
  • flexible linkers between domains
  • can be put into different orders
  • wetlab:
    • goal: library of physical DNA of single modules to be able to put into different orders
    • start with tyrocidine pathway
  • software:
  • tell what you want
  • has info on different pathways and domains
  • tells me how to ligate everything together etc
  • specifity is dependent on CA domain
  • look at what is most probable to work later

--> calculate evolutionary distance according to the taxon ids in the database

  • 4 nodes:
    • b.brevis
    • e.coli.
    • p. aeruginosa
    • b.subtilis
  • Discussion about Linkers:
    • should they be standardized?
    • how should they look like?
    • domains are highly conserved among species and linkers are NOT conserved at all

Wiki-Design

  • responsible persons?? Anja, Tania
  • A brainstorming session would be good
  • we are sure that wwe want it simple but the motto philosopher stone
  • explain logo on the wiki
ideas
  • every project has ONE object from the logo
  • Höhlenmalerei was an idea
  • maybe two possible ways to get to the wiki

software NRPS

  • visualize an overview of project (structure)
  • what is already done?
  • what is to do?
  • linker discussion
  • there are more than 500 monomer
  • one module consists in different domains

Agenda for the next meeting (19.07.2013)

  1. Organisation (Hanna)
  2. cooperation with Freiburg (Sophie)
  3. sponsoring
  4. software-NRPS (Konrad & Software)
  5. Lab-work
  6. human practices (slight)

to do

  • wiki design
  • human practices
  • organisation of team:
    • list of protocol writers,
    • for every detail there has to be defined a responsible person
  • doodle for concept day

Organisation

Hanna is new in the team as an advisor and will support us during the project. We have to book hotel and train tickets for her.

Freiburg-Cooperation

Three Freiburg team mmbers have been to Heidelberg monday/tuesday for cooperation. We did a Gibson Assembly with their constructs and transformed e. coli via electroporation and chemical transformation. After having dinnner they left the next day.

Sponsoring

  • Klaus-Tschira-Stiftung supports us
  • eppendorf supports us with consumables and hardware to lend

NRPS-Software

plan for web-interface and software/database-structure

  • webinterface is ready to feed the database with details to single NRPSs; we will provide the software with about 10-20 NRPS pathways.
  • getting the single domains is time consuming. automatization?
  • -> tool to connect Pfam, clustalO and NCBI to automatically get domain borders for the databas!

Lab

  • delftibacin
    • DelH is amplified; cloning using gibson approach after reassemlby of backbone
    • Del-rest: single parts are missing
  • indigoidine
    • first Streptomyces strain didn't carry our gene of interest; new strain arrived today
    • construct with exchangeable PPTases (gibson or restriction)
  • tyrocidine
    • module shuffling to test concept of modularity
    • bacterial strain is there, primer will be there next week
    • almost no restriction sites for RFC25

milestones

  • DelH is few days in delay, backbone causes trouble
  • DelRest is somewhat in plan
  • Indigoidine is two weeks in delay but no other project part is influenced since we decided to start the tyrocidine part in parallel
  • Tyrocidine; few days in delay because of primer shipment
  • iGEM42; part time work is sufficient as expected
  • NRPS-library; milestones need to be set; until now in time
  • genomic integration; back-up strategy to clone the methylmalonyl-CoA-pathway on a plasmid to validate our delftibactin-e.coli-strain

human practise

  • panel discussion
  • life science lab, schools, essay on gold recovery
  • discuss with NGOs about gold recovery, ecology and economy
  • regional small jewelries, maybe for sponsoring
  • interaction with lab-art project in summer
  • publications in systembiologie.de, RNZ, Uni-Zeitung, ...
  • don't put too much time in human practise

next meeting

with supervisors: tuesday July 30th; 5.30 pm

General organisation stuff

  • Fridge should be defrosted -> does not close to well right now
  • Keep an eye on backup bottles of agarose, buffers, etc. and discard gel remains....
  • Handle ethidiumbromide with care and do use new gloves for touching other stuff as buffers for instance; close gel chambers; better communication and more chambers needed
  • Sign in to the cycler calendars
  • Mini prep: more efficient with 2 min lysis
  • Write on autolcavation stuff: who, content, date
  • Store autoclaved PCR tubes in the cupboard over the sink
    • General duties lab service: tips, autoclavation waste, generel order, plates of all resistances stored, consumables -> Hanna will prepare a short to do list
  • Keep door to preparation room shut (noise)
  • Friday at 10 am weekly team meeting for general issues (advisor meeting on tuesday and thursday 8.30 am)
  • Further team building events (e.g. barbecues) should be planned
  • Karl-Steinbruch scholarship won't be pursued further; Additional discussion with Roland Eils at the next meeting
  • Dispute disagreements with other persons directly
  • Google calendars: keep lab times updated!
  • Plasmid cards: we need them of each subproject; Nomenclature: as primers only with 'p' prefix; ask others if you do not know how to prepare them
  • Do not forget the wiki documentation
  • Abstract, tracks etc. have to be submitted until beginning of august -> has to be communicated with Roland, too. Project titles, abstracts etc. can be changed until later deadline. => Hanna, Fanny and Tania will prepare some drafts.
  • Biosafety fonds -> Philipp

Design

  • Invite Lang & Pflanz again? Ask Roland.
  • T-shirts -> polo, embroidery, color: dark blue, black or light green, light blue -> proposals will be prepared by Tania for next meeting
  • wiki design:
    • different languages: german (all), english(all), italian (Barbara), portugese(Tania), russian(Ilia), france (Philipp), spanish (Hanna), greek (Nikos), ukrain (Fanny), chinese (Philipp), japanese(Ralf), vietnamese(Fanny), hebrew (Tania), klingon(Ilia), korean (Jin)
    • chart of colour range?
    • Tania and Anja present their Wiki layout proposals -> Web-implementation by Joshua and Nils when layout graphics design terminated

Human practices

- Art, panel discussion (synthetic foods), schools (are interested, Tania will talk to the teachers) (-> as backup)

Judging criteria

  • no dates online yet
  • we have to select three tracks -> which ones?
  • our favorites (until now):

- Manufacturing - foundational Advance - environment

  • project description can be adjusted until wiki freeze (but is no judging criteria): make it more lurid (abstract as well) and integrate synthetic biology better

Freiburg constructs

  • characterize constructs? => Help another Team criteria
  • but these are eucaryotic constructs
  • check on different cell lines, media and digests
  • critical because incubator could be contaminated by us -> disastrous for Di Ventura group
  • we have no cell culture -> send cell lines to Freiburg instead
  • we have to fulfill one of three gold medal requirements (Parts debugging, characterize other parts, essay on safety/environment/effects of proeject)
  • characterize SFP? Has not been submitted -> cannot do it -> re-submit pptases
  • essay backup solution

Plan for Tomorrow (Meeting with Roland Eils)

  • Milestones
  • Short lab update
    • Indigoidine [as a new reporter system; try all suggested T-domains; show benefits of software for Indigoidine project]
    • Software [set up criteria, which describe similarity of modules, e.g. by literature research, Nikos will ask a member of König group, who worked with t-domains, for help]
    • Tyrocidine [detection: Wombacher group; in-vitro/in-vivo in parallel; maybe his-tag?; HPLC before? -> enrichment of fragments; CPEC as alternative for Gibson (worked for Ralf); e. coli lysate (e.g. in-vitro translation); do some literature research on how other groups detected NRPs; non-proteinogenic amino acids for labeling; concentration gradients native <-> transformed; contact Mr. Lehmann]
    • Del H [wait for results (tomorrow)]
    • Del Rest [waiting for digestion results; Have all fragments, but F-G is too yet]
    • Software [meeting tomorrow before Eils meeting? Present our scheme of the last meeting + slides of subteams]
    • send all slides to Philipp until 3.30 p.m. tomorrow

Sponsoring

  • Böhringer Ingelheim sponsors us with 5.000 €
  • a Gold Smith from Heidelberg donates us 50 €

Wiki-Design & T-shirts

  • we decided to stich our logo on the shirts
  • color: navy
  • golden border around the logo with blue background

Lab update

Indigiodine
  • we have 3 constructs which work
  • now we want to find out which T-domains are the best to use for the NRPS library
  • we are going to clone indigoidine into igem-standard and characterize it
Tyrocidine
  • a few fragments don't have sufficient concentration --> optimization
  • amplification of some fragments didn't work
  • first gibson assembly will be done this week
  • talked to different people because of using their mass-spectrometry
DelH
  • changed the backbone
  • managed to create constructs, did the electroporation
  • there were no colonies growing on the plates
DelRest
  • were able to amplify all parts
  • started to validate fragments
  • start gibson as soon as we checked fragments

NRPS-Software

  • Database is more or less implemented
  • main programm is able to most of what it is supposed to
  • Webinterface has been started but doesn't comprise all features
  • connection Pfam works

Art Cooperation

  • maybe involving artists into our meeting
  • talk to them about our projects --> maybe they like some parts of our projects and we can collaborate
  • we could give them a insight into synthetic biology
  • we can think about offering some of the artists to stay with us during the period of the art project

Organisation

  • Slides until Wednesday, 11 am to Philipp
  • Slides will also go to advisors
  • bring two laptops with working Skype
  • will not talk about safety to Eils
  • BioBricks have to arrive at the registry until September 18

Software

  • C++ part builds almost fully statically
  • database finished, first entries
  • need to verify iGEM requirements for software submission
  • use same design and color palette for all web sites
  • iGEM42, NRPSDesigner will be linked to in the wiki

Del

  • Prof. Eils knows about strains, company's progress

Tyrocidine

  • assembled constructs
  • picked colonies, miniPreps
  • planned: quantification gel -> restriction digest
  • short peptide NRPS in RFC10
  • individual modules for submission planned in RFC10

Indigoidine

  • assembly of parts for submission in progress
  • T-domains shuffling in progress
  • blue colonies with different PPTases (constructs not verified yet)
  • use RFC10 for submission

Thanks to

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