Team:Brown-Stanford/Lab/Notebook/Week5

• REGObricks
  Intro --------------------- ISRU --------------------- Biocement --------------------- S. pasteurii --------------------- Balloon
  Flights --------------------- Transforming --------------------- Biobrick²
• PowerCell
  Intro --------------------- Cyanobacteria --------------------- Mars --------------------- Nutrients
• FRETcetera
  Intro --------------------- FRET etc. --------------------- Construct
• Lab
  Team --------------------- Parts --------------------- Notebook --------------------- Protocols --------------------- Safety
• Partners
  Collab --------------------- Sponsors
• Outreach
  SB5.0 --------------------- NASA --------------------- Lunar Science --------------------- Maker Faire --------------------- BBC --------------------- CBricks --------------------- Ethics --------------------- Regional
  Jamboree

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