Team:Wisconsin-Madison/notebook/Sarah

From 2010.igem.org

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'''Plans for next week'''
'''Plans for next week'''
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=Progress Report 8-6-2010=
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'''Major Accomplishments'''
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'''Plans for next week'''
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----
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=Progress Report 8-13-2010=
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'''Major Accomplishments'''
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'''Plans for next week'''
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----
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=Progress Report 8-20-2010=
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'''Major Accomplishments'''
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'''Plans for next week'''
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----
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=Progress Report 8-27-2010=
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'''Major Accomplishments'''
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'''Plans for next week'''
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----
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=Progress Report 9-3-2010=
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'''Major Accomplishments'''
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'''Plans for next week'''
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Revision as of 04:30, 27 September 2010

Contents

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

(view notebook 6/3-6/10)

Major Accomplishments

  • 1/2 clones

RcsB:pBAD18BB:DH10b colony PCR

9.jpg

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

(view notebook 6/11-6/18)

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

Plans for next week

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


Retrieved from "http://2010.igem.org/Team:Wisconsin-Madison/notebook/Sarah"