Team:Wisconsin-Madison/notebook/Sarah

From 2010.igem.org

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=Progress Report 6-3-2010=
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==Progress Report 6-3-2010==
-
 
+
'''Major Accomplishments'''
'''Major Accomplishments'''
* pBAD's: 18, 33, 35 were successfully made into biobrick vectors
* pBAD's: 18, 33, 35 were successfully made into biobrick vectors
Line 24: Line 23:
* Research has been mapped and divided
* Research has been mapped and divided
* ordered parts have either been confirmed or marked as incorrect
* ordered parts have either been confirmed or marked as incorrect
-
 
'''Plans for next week'''
'''Plans for next week'''
* Finish encapsulation clones
* Finish encapsulation clones
Line 31: Line 29:
* Finnish individual research and have team meeting
* Finnish individual research and have team meeting
* inducible/repressable promotion system
* inducible/repressable promotion system
-
 
-
 
-
----
 
-
 
''Day by Day overview:''
''Day by Day overview:''
-
 
'''5-24-2010'''  
'''5-24-2010'''  
* Lab materials, organization
* Lab materials, organization
Line 71: Line 64:
* Planned 4 testing clones of encapsulation
* Planned 4 testing clones of encapsulation
* Encapsulation clones: digestion, extraction, overnight ligation  
* Encapsulation clones: digestion, extraction, overnight ligation  
-
 
+
<br>
-
 
+
==Progress Report 6-11-2010==
-
=Progress Report 6-11-2010=
+
-
(view notebook 6/3-6/10)
+
-
 
+
'''Major Accomplishments'''
'''Major Accomplishments'''
* 1/2 clones  
* 1/2 clones  
RcsB:pBAD18BB:DH10b colony PCR
RcsB:pBAD18BB:DH10b colony PCR
-
 
[[File: 9.jpg| 400px]]
[[File: 9.jpg| 400px]]
-
 
'''Plans for next week'''
'''Plans for next week'''
* Acid survival tests (growth curves and acid dunk)
* Acid survival tests (growth curves and acid dunk)
Line 89: Line 77:
* alkaline lysis and digestion of C3 of above colony PCR
* alkaline lysis and digestion of C3 of above colony PCR
-
----
 
'''6-3-2010'''
'''6-3-2010'''
* Got out of Jury Duty
* Got out of Jury Duty
Line 95: Line 82:
* Map of LacZ - wrong part
* Map of LacZ - wrong part
* Liquid Cultures
* Liquid Cultures
-
 
'''6-4-2010'''
'''6-4-2010'''
* Map (no cut) of pBAD clones
* Map (no cut) of pBAD clones
* encapsulation clones: Miniprep, digestion, gel extraction (wrong)
* encapsulation clones: Miniprep, digestion, gel extraction (wrong)
-
 
'''6-7-2010'''
'''6-7-2010'''
* advisor meeting
* advisor meeting
* encapsulation clones to ligation
* encapsulation clones to ligation
-
 
'''6-8-2010'''
'''6-8-2010'''
* transformation and plating of encapsulation clones
* transformation and plating of encapsulation clones
-
 
'''6-9-2010'''
'''6-9-2010'''
* transformation and plating of encryption clones
* transformation and plating of encryption clones
-
 
'''6-10-2010'''
'''6-10-2010'''
* screening of pBAD clones - colony PCR
* screening of pBAD clones - colony PCR
* encapsulation research
* encapsulation research
-
 
+
<br>
-
=Progress Report 6-18-2010=
+
==Progress Report 6-18-2010==
-
(view notebook 6/11-6/18)
+
-
 
+
'''Major Accomplishments'''
'''Major Accomplishments'''
* Identified and verified next set of parts for cloning
* Identified and verified next set of parts for cloning
-
 
'''Plans for next week'''
'''Plans for next week'''
* screen more clones (YgiV and RfaI)
* screen more clones (YgiV and RfaI)
Line 126: Line 105:
* pBAD series + RFP
* pBAD series + RFP
* YgiV and RfaI in pBADs
* YgiV and RfaI in pBADs
-
 
----
----
'''6/11/10'''
'''6/11/10'''
Line 132: Line 110:
* Sequencing rxn of pBADBB [18,33,35]
* Sequencing rxn of pBADBB [18,33,35]
* Planned Map of new ordered parts
* Planned Map of new ordered parts
-
 
'''6/14/10'''
'''6/14/10'''
* Mapping of new plates of biobrick vector pBADs
* Mapping of new plates of biobrick vector pBADs
* primer Design of 6 new primers for two well characterized encapsulation genes
* primer Design of 6 new primers for two well characterized encapsulation genes
* Restriction Map of 9 new parts - all correct
* Restriction Map of 9 new parts - all correct
-
 
'''6/15/10'''
'''6/15/10'''
* Miniprep of new parts, rcsB clone, and pBADBBs
* Miniprep of new parts, rcsB clone, and pBADBBs
* Restriction map of more parts - ??
* Restriction map of more parts - ??
-
 
'''6/16/10'''
'''6/16/10'''
* Ligation of RfaI:pBAD35BB
* Ligation of RfaI:pBAD35BB
* Website and Logo
* Website and Logo
-
 
'''6/17/10'''
'''6/17/10'''
* Restriction map of iffy parts
* Restriction map of iffy parts
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* Ran mary's gel with 2x concentration of DNA
* Ran mary's gel with 2x concentration of DNA
* ReDigestion of pBAD35BB
* ReDigestion of pBAD35BB
-
 
'''6/18/10'''
'''6/18/10'''
* colony PCR of RfaI:pBAD15BB:DH10B x10- fail
* colony PCR of RfaI:pBAD15BB:DH10B x10- fail
* ReDigest and extract pBAD35BB
* ReDigest and extract pBAD35BB
-
 
+
<br>
-
=Progress Report 6-25-2010=
+
==Progress Report 6-25-2010==
-
 
+
'''Major Accomplishments'''
'''Major Accomplishments'''
* Discovered flaw in pBAD35BB:  
* Discovered flaw in pBAD35BB:  
Line 168: Line 140:
** '''Q:''' Do you know anything about a point mutation your lab did or why this is? (DamI methylation?)  
** '''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: [https://mywebspace.wisc.edu/sandock/iDIET%20Project.pdf Project Plan]
* New and complete planning of cloning, experiments and parts: [https://mywebspace.wisc.edu/sandock/iDIET%20Project.pdf Project Plan]
-
 
-
'''Plans for next week'''
 
-
*
 
-
 
----
----
'''6-20-2010'''
'''6-20-2010'''
Line 177: Line 145:
** 35 has inactive PstI
** 35 has inactive PstI
** 33 has inactive extra EcoRI
** 33 has inactive extra EcoRI
-
 
'''6-21-2010'''
'''6-21-2010'''
* Alkaline Lysis and Digestion of RcsB:pBAD18BB - WRONG
* Alkaline Lysis and Digestion of RcsB:pBAD18BB - WRONG
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* confirmation of faulty 35 PstI from sequencing data
* confirmation of faulty 35 PstI from sequencing data
* New digestion protocol uses less reagents
* New digestion protocol uses less reagents
-
 
'''6-22-2010'''
'''6-22-2010'''
* pBAD35 - around the world PCR, DpnI digest, PCR clean up, XbaI digest, PCR cleanup, Ligation, transformation, plating
* 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
* colony PCR of direct genes: wz, wzT, yjb, yjbT
-
 
'''6-23-2010'''
'''6-23-2010'''
* got 3 genes from colony PCR of direct genes (not yjb)
* 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
* pBAD34 - around the world PCR, DpnI digest, PCR clean up, XbaI digest, PCR cleanup, Ligation, transformation, plating
* Phussion PCR from colony PCR template
* Phussion PCR from colony PCR template
-
 
'''6-24-2010'''
'''6-24-2010'''
* Finished organizing and typing project plan
* Finished organizing and typing project plan
Line 201: Line 165:
** [[File:222.jpg]]
** [[File:222.jpg]]
* Ran PCR off of colony PCR as Template - FAIL (use colony PCR next time)
* Ran PCR off of colony PCR as Template - FAIL (use colony PCR next time)
-
 
'''6-25-2010'''
'''6-25-2010'''
* Alkaline lysis of pBAD35BB and pBAD34BB
* Alkaline lysis of pBAD35BB and pBAD34BB
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** [[File:222.jpg]]
** [[File:222.jpg]]
* Initial digestion of and pBAD34BB - Fail
* Initial digestion of and pBAD34BB - Fail
-
 
+
<br>
-
=Progress Report 7-2-2010=
+
==Progress Report 7-2-2010==
-
 
+
'''Major Accomplishments'''
'''Major Accomplishments'''
* Decided the pBAD vectors were a lost cause
* Decided the pBAD vectors were a lost cause
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* Clones assigned to team
* Clones assigned to team
* Created clones box and updated completed parts
* Created clones box and updated completed parts
-
 
'''Plans for next week'''
'''Plans for next week'''
* Clone
* Clone
* New LuxR Rev primer that includes stop codon
* New LuxR Rev primer that includes stop codon
-
 
----
----
'''6-28-2010'''
'''6-28-2010'''
* cloning of RcsA downstream of RcsB in iGEM vector pSB1AK3
* cloning of RcsA downstream of RcsB in iGEM vector pSB1AK3
-
 
'''6-29-2010'''
'''6-29-2010'''
* Successful cloning of RcsB+RcsA - Colony PCR
* Successful cloning of RcsB+RcsA - Colony PCR
Line 235: Line 194:
* cloning of YgiV downstream of pLacI+RBS in iGEM vector pSB1AK3
* cloning of YgiV downstream of pLacI+RBS in iGEM vector pSB1AK3
* cloning of RfaI downstream of pLacI+RBS in iGEM vector pSB1AK3
* cloning of RfaI downstream of pLacI+RBS in iGEM vector pSB1AK3
-
 
'''6-30-2010'''
'''6-30-2010'''
* pBAD vectors have given us too much trouble. Time to move on.
* pBAD vectors have given us too much trouble. Time to move on.
Line 244: Line 202:
** Correct usage of ribosome binding sites
** Correct usage of ribosome binding sites
* Ordered and transformed all nessesary parts from registry
* Ordered and transformed all nessesary parts from registry
-
 
'''7-1-2010'''
'''7-1-2010'''
* Finished new project plan
* Finished new project plan
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** [[File:plry.jpg|400px]]
** [[File:plry.jpg|400px]]
* Liquid cultures of transformed parts and successful clones
* Liquid cultures of transformed parts and successful clones
-
 
'''7-2-2010'''
'''7-2-2010'''
* Miniprep of 7 Parts from Registry
* Miniprep of 7 Parts from Registry
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* Cloning of I13507 (RBS+mRFP+TT) downstream of R0065 (InducibleRepressiblePromoter) 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.
** for both clonings, backbones cut but inserts were undigested.
-
 
+
<br>
-
=Progress Report 7-9-2010=
+
==Progress Report 7-9-2010==
-
 
+
'''Major Accomplishments'''
'''Major Accomplishments'''
* Clone: K200021+C0051
* Clone: K200021+C0051
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**[[File:oiue2.jpg|200px]]
**[[File:oiue2.jpg|200px]]
* Sequencing rxn of clones 1 and 2
* Sequencing rxn of clones 1 and 2
-
 
'''7-6-2010'''
'''7-6-2010'''
* Digestion
* Digestion
* Magnetic Bead cleanup and submission for sequencing
* Magnetic Bead cleanup and submission for sequencing
-
 
'''7-7-2010'''
'''7-7-2010'''
*  Cloning of C0051 (Repressor and Deg Tag) downstream of K200021(LacI promoter + RBS) in iGEM vector pSB1AK3
*  Cloning of C0051 (Repressor and Deg Tag) downstream of K200021(LacI promoter + RBS) in iGEM vector pSB1AK3
-
 
'''7-8-2010'''
'''7-8-2010'''
* Colony PCR of clone from yesterday
* Colony PCR of clone from yesterday
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* Liquid culture colony#5, colony #6, K112808, I13507
* Liquid culture colony#5, colony #6, K112808, I13507
* Went to CS for programing help
* Went to CS for programing help
-
 
'''7-9-2010'''
'''7-9-2010'''
* Alkaline Lysis of colony#5, colony#6, K112808, I13507
* Alkaline Lysis of colony#5, colony#6, K112808, I13507
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* Kit Miniprep of colony#6
* Kit Miniprep of colony#6
* Cloning of above clone in front of B0034(RBS) and B0015(TT) - Digestion, Ligation
* Cloning of above clone in front of B0034(RBS) and B0015(TT) - Digestion, Ligation
-
 
+
<br>
-
=Progress Report 7-16-2010=
+
==Progress Report 7-16-2010==
-
 
+
'''Major Accomplishments'''
'''Major Accomplishments'''
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-
 
+
<br>
-
=Progress Report 7-23-2010=
+
==Progress Report 7-23-2010==
-
 
+
'''Major Accomplishments'''
'''Major Accomplishments'''
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-
 
+
<br>
-
=Progress Report 7-30-2010=
+
==Progress Report 7-30-2010==
-
 
+
'''Major Accomplishments'''
'''Major Accomplishments'''
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----
----
-
=Progress Report 8-6-2010=
+
<br>
-
 
+
==Progress Report 8-6-2010==
'''Major Accomplishments'''
'''Major Accomplishments'''
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----
----
-
=Progress Report 8-13-2010=
+
<br>
-
 
+
==Progress Report 8-13-2010==
'''Major Accomplishments'''
'''Major Accomplishments'''
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----
----
-
=Progress Report 8-20-2010=
+
<br>
-
 
+
==Progress Report 8-20-2010==
'''Major Accomplishments'''
'''Major Accomplishments'''
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----
----
-
=Progress Report 8-27-2010=
+
<br>
-
 
+
==Progress Report 8-27-2010==
'''Major Accomplishments'''
'''Major Accomplishments'''
'''Plans for next week'''
'''Plans for next week'''
-
 
-
 
----
----
-
=Progress Report 9-3-2010=
+
<br>
-
 
+
==Progress Report 9-3-2010==
'''Major Accomplishments'''
'''Major Accomplishments'''
'''Plans for next week'''
'''Plans for next week'''
-
 
-
 
----
----

Revision as of 02:54, 30 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

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

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

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