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From 2010.igem.org

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Revision as of 04:28, 27 September 2010

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

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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

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

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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

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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

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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

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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

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7-5-2010

• Digestion of K112808 and I13507
• 2nd Proof-Digestion: clones 1 and 2
  • 200px
• 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

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Progress Report 7-23-2010

Major Accomplishments

Plans for next week

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Progress Report 7-30-2010

Major Accomplishments

Plans for next week

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