"
Team:Wisconsin-Madison/notebook/Sarah
From 2010.igem.org
Progress Report 6-3-2010
Major Accomplishments
- pBAD's: 18, 33, 35 were successfully made into biobrick vectors
- Primers are done and being ordered
- Lab is organized and a method of labeling and storage is established
- Research has been mapped and divided
- ordered parts have either been confirmed or marked as incorrect
Plans for next week
- Finish encapsulation clones
- Test encapsulation clones (pH and growth curves in testing strain)
- comp cells with either MG1655 or BL21
- Finnish individual research and have team meeting
- inducible/repressable promotion system
Day by Day overview: 5-24-2010
- Lab materials, organization
- Comp Cell overview
- Began Primer Deisgn
- pBAD point mutations
5-25-2010
- began around the world cloning of pBADs up to Digestion overnight
- Primer design
- Redid PCR of pBAD34
5-26-2010
- pBAD 34: digestion to overnight ligation
- pBAD 18,33,35: Ligating to plating transformation
- Finished primer design
5-27-2010
- pBAD34: transformed and plated
- pBAD 18,33,35: picked colonies to screen
- restriction mapping of parts ordered
- Cleaned out fridge and freeze and organized by project (lac v encypt)
5-28-2010
- Alkaline lysis of 23 colonies
- Restriction mapping of pBAD-BB 18, 34, 35 - SUCCESSFUL
- Liquid cultures of pBAD34
- Double digest of ordered parts – Lactose: 3 capsule genes OK
5-29-2010
- Alkaline lysis for screening of pBAD34-BB
6-1-2010
- restriction map of pBAD34-BB – Fail
- inventor of correct vs wrong parts
- planned clones for next day
- research on project
6-2-2010
- Mapped out needed research and assigned to team
- Planned 4 testing clones of encapsulation
- Encapsulation clones: digestion, extraction, overnight ligation
Progress Report 6-11-2010
Major Accomplishments
- 1/2 clones
RcsB:pBAD18BB:DH10b colony PCR
Plans for next week
- Acid survival tests (growth curves and acid dunk)
- pH promoter (RFP and error prone PCR)
- get encapsulation clones
- more encapsulation research
- alkaline lysis and digestion of C3 of above colony PCR
6-3-2010
- Got out of Jury Duty
- Made silent point mutation primers for pBAD33 (extra EcoRI)
- Map of LacZ - wrong part
- Liquid Cultures
6-4-2010
- Map (no cut) of pBAD clones
- encapsulation clones: Miniprep, digestion, gel extraction (wrong)
6-7-2010
- advisor meeting
- encapsulation clones to ligation
6-8-2010
- transformation and plating of encapsulation clones
6-9-2010
- transformation and plating of encryption clones
6-10-2010
- screening of pBAD clones - colony PCR
- encapsulation research
Progress Report 6-18-2010
Major Accomplishments
- Identified and verified next set of parts for cloning
Plans for next week
- screen more clones (YgiV and RfaI)
- pt mutations of 18 and 33
- pH promoter + RFP
- pBAD series + RFP
- YgiV and RfaI in pBADs
6/11/10
- Colony PCR pH promoter+RFP:pBAD35:DH10b
- Sequencing rxn of pBADBB [18,33,35]
- Planned Map of new ordered parts
6/14/10
- Mapping of new plates of biobrick vector pBADs
- primer Design of 6 new primers for two well characterized encapsulation genes
- Restriction Map of 9 new parts - all correct
6/15/10
- Miniprep of new parts, rcsB clone, and pBADBBs
- Restriction map of more parts - ??
6/16/10
- Ligation of RfaI:pBAD35BB
- Website and Logo
6/17/10
- Restriction map of iffy parts
- Tranformation and plating of RfaI:pBAD35BB:DH10b
- PCR, Digestion, cleanup of RFP for pBAD characterization - fail after cleanup
- Ran mary's gel with 2x concentration of DNA
- ReDigestion of pBAD35BB
6/18/10
- colony PCR of RfaI:pBAD15BB:DH10B x10- fail
- ReDigest and extract pBAD35BB
Progress Report 6-25-2010
Major Accomplishments
- Discovered flaw in pBAD35BB:
- PstI cut site in inactive
- Sequencing data shows that the PstI cutsite is missing a single basepair
- Completed new pBAD35BB
- No point mutation is required for pBAD33BB:
- Extra EcoRI cut site in inactive in both the original 33 and our biobrick version
- Q: Do you know anything about a point mutation your lab did or why this is? (DamI methylation?)
- New and complete planning of cloning, experiments and parts: Project Plan
6-20-2010
- EcoRI, XbaI, SpeI, PstI digestion of pBADBB 18, 33, 35
- 35 has inactive PstI
- 33 has inactive extra EcoRI
6-21-2010
- Alkaline Lysis and Digestion of RcsB:pBAD18BB - WRONG
- this is odd: colony PCR said otherwise
- confirmation digestion of 33BB and original and 35 from yesterday
- confirmation of faulty 35 PstI from sequencing data
- New digestion protocol uses less reagents
6-22-2010
- pBAD35 - around the world PCR, DpnI digest, PCR clean up, XbaI digest, PCR cleanup, Ligation, transformation, plating
- colony PCR of direct genes: wz, wzT, yjb, yjbT
6-23-2010
- got 3 genes from colony PCR of direct genes (not yjb)
- pBAD34 - around the world PCR, DpnI digest, PCR clean up, XbaI digest, PCR cleanup, Ligation, transformation, plating
- Phussion PCR from colony PCR template
6-24-2010
- Finished organizing and typing project plan
- Liquid Cultures of pBAD35BB and pBAD34BB
- Initial digestion of pBAD35BB - clones successful
- SpeI cuts clones once and no cuts on originals
- File:222.jpg
- Ran PCR off of colony PCR as Template - FAIL (use colony PCR next time)
6-25-2010
- Alkaline lysis of pBAD35BB and pBAD34BB
- Secondary digestion of pBAD35BB - Successful
- CLONES - BBcutsits=once, KpnI=no cut
- ORIGINAL - KpnI=once
- File:222.jpg
- Initial digestion of and pBAD34BB - Fail
Progress Report 7-2-2010
Major Accomplishments
- Decided the pBAD vectors were a lost cause
- New Project Plan
- 2 clones RcsB+RcsA and pLacI+RBS+YgiV on my first two tries without pBAD vectors
- Clones assigned to team
- Created clones box and updated completed parts
Plans for next week
- Clone
- New LuxR Rev primer that includes stop codon
6-28-2010
- cloning of RcsA downstream of RcsB in iGEM vector pSB1AK3
6-29-2010
- Successful cloning of RcsB+RcsA - Colony PCR
- 400px
- columns 2 and 4 show correct banding for B+A
- columns 1 and 3 show correct banding for B
- caps in PCR were not tight and 5-12 became dehydrated
- cloning of YgiV downstream of pLacI+RBS in iGEM vector pSB1AK3
- cloning of RfaI downstream of pLacI+RBS in iGEM vector pSB1AK3
6-30-2010
- pBAD vectors have given us too much trouble. Time to move on.
- New project plan
- Project Plan
- No pBAD vectors
- Better incorporation of parts offered by registry
- Correct usage of ribosome binding sites
- Ordered and transformed all nessesary parts from registry
7-1-2010
- Finished new project plan
- Colony PCR of cloning from 6/29
- Success - pLacI+RBS+YgiV - bottom ~700bp
- Fail - pLacI+RBS+RfaI - top ~1300bp
- 400px
- Liquid cultures of transformed parts and successful clones
7-2-2010
- Miniprep of 7 Parts from Registry
- Cloning of K112808 (lysis cassette + T) downstream of B0034 (RBS) in iGEM vector pSB1A2
- Cloning of I13507 (RBS+mRFP+TT) downstream of R0065 (InducibleRepressiblePromoter) in iGEM vector pSB1A2
- for both clonings, backbones cut but inserts were undigested.
Progress Report 7-9-2010
Major Accomplishments
- Clone: K200021+C0051
- wrong parts: K112808 and I13507 (both important parts)
- All code for wiki done, just need to fill it in and link to home
Plans for next week
- clone
7-5-2010
- Digestion of K112808 and I13507
- 2nd Proof-Digestion: clones 1 and 2
- Sequencing rxn of clones 1 and 2
7-6-2010
- Digestion
- Magnetic Bead cleanup and submission for sequencing
7-7-2010
- Cloning of C0051 (Repressor and Deg Tag) downstream of K200021(LacI promoter + RBS) in iGEM vector pSB1AK3
7-8-2010
- Colony PCR of clone from yesterday
- 200px
- Band = 825 + 144 = 970
- Liquid culture colony#5, colony #6, K112808, I13507
- Went to CS for programing help
7-9-2010
- Alkaline Lysis of colony#5, colony#6, K112808, I13507
- Double digest of above parts
- 2nd proof-digest of above clones
- Kit Miniprep of colony#6
- Cloning of above clone in front of B0034(RBS) and B0015(TT) - Digestion, Ligation
Progress Report 7-16-2010
Major Accomplishments
Plans for next week
Progress Report 7-23-2010
Major Accomplishments
Plans for next week
Progress Report 7-30-2010
Major Accomplishments
Plans for next week
Progress Report 8-6-2010
Major Accomplishments
Plans for next week
Progress Report 8-13-2010
Major Accomplishments
Plans for next week
Progress Report 8-20-2010
Major Accomplishments
Plans for next week
Progress Report 8-27-2010
Major Accomplishments
Plans for next week
Progress Report 9-3-2010
Major Accomplishments
Plans for next week
