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:
- Indigoidine (closed): Rald, Konrad, Nikos
- Del H lab protocol/methods (closed) : Hanna, Fanny, Sophie
- Del Rest (A to P) (closed) : Flo, Ilia, Nikos, Nils
- NRPS : Tania, Joschua, Ilia
- 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
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
- 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
- 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)
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
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
- 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)
- Template mail for sponsoring is nearly ready
- Template project proposal of Eils didn't arrive yet
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