Team:Brown-Stanford/Lab/Notebook/Week5

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== ''' July 11, 2011''' ==
 +
===PowerCell===
 +
*Transformation of GFPmut3B into TOP10 went okay,
 +
*Made liquid culture in LB + amp (100 μg/ml)
 +
*4ml BG11, 1ml from stock culture
 +
*Made more working stocks of cyanos
 +
*Rehydrated CscB into 20ul TE; transformed TOP10 cells; PCR with Gibson primers (two PCRs: RBS|CscB, CscB|TTSP)
 +
*transformed 100ul TOP10 cells with 2ul of hydrated CscB; x2 replicates; 25min on ice, 2min heat shock @ 42C, then add 500ul LB and place in 37C for ~20min
 +
*two plates in 37C at 5:30pm
 +
*Viewed plates at 9:30am, 100s of colonies; placed in 4C in Rm335
 +
 +
*Make antibiotic plates (jov/lei),
 +
*Kanamycin 40 ug/mL
 +
*ampicillin 100 ug/mL
 +
*tetracycline 10 ug/mL
 +
*need amp/tet, amp, kan
 +
*Design primers for veg promoter + GFP-only constructs
 +
*PCR amplfication anabaena vegetative promoter-mid RBS
 +
*PCR cleanup on Nostoc promoter--resulted in 25.8ng/ul of dna product, some fifteen μl
 +
*micrographs using oil immersion, get condenser-optimized pictures of cyanos
 +
*(*synechocystis 6803: need real picture of cells; cyanothece 7822 needs denser sample; anabaena 7120 needs non-airbubble)
 +
== ''' July 12, 2011''' ==
 +
 +
===PowerCell===
 +
*work division: Eli, Julius, Evan,  yet to do, done
 +
*imaged PCRs from 7/11: two cscB rxns (the RBS-cscB part, and the cscB-TTSP part); Anabaena promoter-mid PCR as well
 +
*miniprep’d the GFPmut3b out of transformant TOP10 liquid cultures.  Plasmids kept in tray with norman’s plasmids in 335 4?C.
 +
*streaked out Elhai and Norman plasmids
 +
*Norman plasmids: 100ug/ml for pRL1383a
 +
*cscB liquid culture in preparation for plasmid harvest on 7/13
 +
*innoculated 2ml of LB amp, 3x replicates; placed in 37C at 5:15pm
 +
*finished cryostock
 +
*PCRs: Anabaena promoter+weak/strong RBS; Nostoc weak/strong RBS; GFPmut3b with ends
 +
==FRETSensor==
 +
*Amp and kan plates made
 +
 +
== ''' July 12, 2011''' ==
 +
===PowerCell===
 +
Listened to Jay Keasling's speech on applying synbio to growing biological organisms.
 +
==FRETSensor==
 +
*Amp/Tet + spec plates made
 +
 +
== ''' July 13, 2011''' ==
 +
 +
===PowerCell===
 +
*image PCRs: Anabaena promoter+weak/strong RBS; Nostoc weak/strong RBS; GFPmut3b with ends.  not much showed up, although bands were present in last lanes (identity?)
 +
*cut out most visible (Ana Strong) for gel extraction.
 +
*plasmid prep cscB liquid cultures from 7/12
 +
*picked colony from conjugal transformation plasmid lines and make antibiotic liquid cultures
 +
===REGOBrickes===
 +
*Made new Bang plates, and resolved the issue with pH. Instead of pHing to 6.8, pHed to 8.5, which helped growth considerably.
 +
*Possible plasmids for transformation of S. pasteurii (from B subtilis protocols)
 +
**pUB110
 +
**pHT43
 +
**pHT 01
 +
*Others, from Keggins et al (uploaded) include
 +
**pPL576
 +
**pPL10
 +
**pPL7065
 +
**pPL2
 +
==FRETSensor==
 +
*SOB made, K12 grown up for competency
 +
 +
== ''' July 14, 2011''' ==
 +
 +
===PowerCell===
 +
*cryostock cargo/helper/conjugative plasmid strain liquid cultures
 +
*gel extraction on Ana strong band cut on 7/13 (nanodrop: <5ng/ml)
 +
*imaged Ana weak, Nos weak/strong (from 7/12 PCR)
 +
*cut out 600bp band of Ana weak 50- (there were two bands, strong @ ~600bp for 50-, very faint around 500bp for all) and ~600bp band for Nos weak (all four temps), gel extraction
 +
*plasmid prep of liquid conjugal transformation plasmid cultures (just the ones we will eventually need to modify); stored @4C in Rm 335, “Norman’s plasmids” rack
 +
*pRL25: 30.4ng/ul
 +
*pRL1383a: 26.4ng/ul
 +
*BBpRL: 16.75ng/ul
 +
*ran big PCR of cscB with two ends, cscB with wk and str RBS’s needed for gibson
 +
 +
===REGOBricks===
 +
*printed out research articles detailing transformation protocols for subtilis that we hope to adapt for pasteurii:
 +
*Digestion to Protoplast, then transformation
 +
*soil transformation
 +
*CaCl<sub>2 </sub>
 +
 +
*features/reasons to use pUB110:
 +
**EcoR1 cut sites
 +
**50 copies/cell
 +
*If we needed to try phage transformation, pUB was a lot more amenable to those protocols than other plamsids.
 +
*Other vectors include two from Cambridge (2008):
 +
*pBSINT1 - B. subtilis integration vector (5632 bp) - inserted in amylase gene
 +
*pBSEP - episomal
 +
==FRETSensor==
 +
*K12 competency protocol done, 45 tubes of cells
 +
 +
== ''' July 15, 2011''' ==
 +
 +
===PowerCell===
 +
*imaged  PCR of cscB with two ends, cscB with wk and str RBS’s (from 7/14)
 +
*visible bands for cscB-TTSP, RBSw cscB and RBSs cscB; cut out bands and ran gel extraction
 +
*imaged Ana mid, positive control and negative control for RBSm-cscB (from PCR done on 7/11)
 +
*cut out Ana med bands, ran gel extraction; also performed PCR cleanup using Qiagen kit (result 5ng/ul for gel ext, 17.5ng/ul for PCR cleanup) to use for 2nd round PCR
 +
*imaged RBS-GFP-TTSP from 7/12, (-) cont for GFP from 7/14;
 +
*cut out four bright bands, NOT YET GEL EXTRACTED; also enough quantity to PCR cleanup directly (NOT YET DONE)
 +
*PCR: second round Ana weak, strong, med, Nostoc weak
 +
*PCR: RBSm|cscB
 +
===REGOBricks===
 +
*Dosier Media
 +
**5g soy protein
 +
**15g peptone from casein
 +
**10 micrograms manganese sulfate
 +
**20 g urea
 +
**5g NaCl
 +
==FRETSensor==
 +
*Competency of K12 tested, GFPmut3b transformation has lots of colonies, negative control is completely empty
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Latest revision as of 18:10, 28 September 2011

Brown-Stanford
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July 11, 2011

PowerCell

  • Transformation of GFPmut3B into TOP10 went okay,
  • Made liquid culture in LB + amp (100 μg/ml)
  • 4ml BG11, 1ml from stock culture
  • Made more working stocks of cyanos
  • Rehydrated CscB into 20ul TE; transformed TOP10 cells; PCR with Gibson primers (two PCRs: RBS|CscB, CscB|TTSP)
  • transformed 100ul TOP10 cells with 2ul of hydrated CscB; x2 replicates; 25min on ice, 2min heat shock @ 42C, then add 500ul LB and place in 37C for ~20min
  • two plates in 37C at 5:30pm
  • Viewed plates at 9:30am, 100s of colonies; placed in 4C in Rm335
  • Make antibiotic plates (jov/lei),
  • Kanamycin 40 ug/mL
  • ampicillin 100 ug/mL
  • tetracycline 10 ug/mL
  • need amp/tet, amp, kan
  • Design primers for veg promoter + GFP-only constructs
  • PCR amplfication anabaena vegetative promoter-mid RBS
  • PCR cleanup on Nostoc promoter--resulted in 25.8ng/ul of dna product, some fifteen μl
  • micrographs using oil immersion, get condenser-optimized pictures of cyanos
  • (*synechocystis 6803: need real picture of cells; cyanothece 7822 needs denser sample; anabaena 7120 needs non-airbubble)

July 12, 2011

PowerCell

  • work division: Eli, Julius, Evan, yet to do, done
  • imaged PCRs from 7/11: two cscB rxns (the RBS-cscB part, and the cscB-TTSP part); Anabaena promoter-mid PCR as well
  • miniprep’d the GFPmut3b out of transformant TOP10 liquid cultures. Plasmids kept in tray with norman’s plasmids in 335 4?C.
  • streaked out Elhai and Norman plasmids
  • Norman plasmids: 100ug/ml for pRL1383a
  • cscB liquid culture in preparation for plasmid harvest on 7/13
  • innoculated 2ml of LB amp, 3x replicates; placed in 37C at 5:15pm
  • finished cryostock
  • PCRs: Anabaena promoter+weak/strong RBS; Nostoc weak/strong RBS; GFPmut3b with ends

FRETSensor

  • Amp and kan plates made

July 12, 2011

PowerCell

Listened to Jay Keasling's speech on applying synbio to growing biological organisms.

FRETSensor

  • Amp/Tet + spec plates made

July 13, 2011

PowerCell

  • image PCRs: Anabaena promoter+weak/strong RBS; Nostoc weak/strong RBS; GFPmut3b with ends. not much showed up, although bands were present in last lanes (identity?)
  • cut out most visible (Ana Strong) for gel extraction.
  • plasmid prep cscB liquid cultures from 7/12
  • picked colony from conjugal transformation plasmid lines and make antibiotic liquid cultures

REGOBrickes

  • Made new Bang plates, and resolved the issue with pH. Instead of pHing to 6.8, pHed to 8.5, which helped growth considerably.
  • Possible plasmids for transformation of S. pasteurii (from B subtilis protocols)
    • pUB110
    • pHT43
    • pHT 01
  • Others, from Keggins et al (uploaded) include
    • pPL576
    • pPL10
    • pPL7065
    • pPL2

FRETSensor

  • SOB made, K12 grown up for competency

July 14, 2011

PowerCell

  • cryostock cargo/helper/conjugative plasmid strain liquid cultures
  • gel extraction on Ana strong band cut on 7/13 (nanodrop: <5ng/ml)
  • imaged Ana weak, Nos weak/strong (from 7/12 PCR)
  • cut out 600bp band of Ana weak 50- (there were two bands, strong @ ~600bp for 50-, very faint around 500bp for all) and ~600bp band for Nos weak (all four temps), gel extraction
  • plasmid prep of liquid conjugal transformation plasmid cultures (just the ones we will eventually need to modify); stored @4C in Rm 335, “Norman’s plasmids” rack
  • pRL25: 30.4ng/ul
  • pRL1383a: 26.4ng/ul
  • BBpRL: 16.75ng/ul
  • ran big PCR of cscB with two ends, cscB with wk and str RBS’s needed for gibson

REGOBricks

  • printed out research articles detailing transformation protocols for subtilis that we hope to adapt for pasteurii:
  • Digestion to Protoplast, then transformation
  • soil transformation
  • CaCl2
  • features/reasons to use pUB110:
    • EcoR1 cut sites
    • 50 copies/cell
  • If we needed to try phage transformation, pUB was a lot more amenable to those protocols than other plamsids.
  • Other vectors include two from Cambridge (2008):
  • pBSINT1 - B. subtilis integration vector (5632 bp) - inserted in amylase gene
  • pBSEP - episomal

FRETSensor

  • K12 competency protocol done, 45 tubes of cells

July 15, 2011

PowerCell

  • imaged PCR of cscB with two ends, cscB with wk and str RBS’s (from 7/14)
  • visible bands for cscB-TTSP, RBSw cscB and RBSs cscB; cut out bands and ran gel extraction
  • imaged Ana mid, positive control and negative control for RBSm-cscB (from PCR done on 7/11)
  • cut out Ana med bands, ran gel extraction; also performed PCR cleanup using Qiagen kit (result 5ng/ul for gel ext, 17.5ng/ul for PCR cleanup) to use for 2nd round PCR
  • imaged RBS-GFP-TTSP from 7/12, (-) cont for GFP from 7/14;
  • cut out four bright bands, NOT YET GEL EXTRACTED; also enough quantity to PCR cleanup directly (NOT YET DONE)
  • PCR: second round Ana weak, strong, med, Nostoc weak
  • PCR: RBSm|cscB

REGOBricks

  • Dosier Media
    • 5g soy protein
    • 15g peptone from casein
    • 10 micrograms manganese sulfate
    • 20 g urea
    • 5g NaCl

FRETSensor

  • Competency of K12 tested, GFPmut3b transformation has lots of colonies, negative control is completely empty

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