Team:Brown-Stanford/Lab/Notebook/Week2

• 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

June 20, 2011

• Made solid, liquid LB
• Made kan, amp solid LB plates
• made CCMB80 Buffer

PowerCell

• Made BG11 Plates
• Streaked out Golden strains on BG11 plates (in the 30? incubator)
• Created 5mL liquid BG11 cultures of all the Golden strains (in the 30? incubator)
• Streaked Norman’s E. coli conjugative plasmid strains on ampicilin to verify plasmid retention

FRETSensor

• Found constructs for fused dockerin-proteins, requested
• Found constructs from Fierobe’s lab for cohesin-cohesin group (Scaf4), requested
• Talked to Dr. Paavola - will have rosettazyme with C. thermocellum cohesin
• Regrowing Evan and Ryan’s strain from Endy’s class

June 21, 2011

• Designed construct (see construct page)
• Designed Co-culture experiment (see experiment design page)
• Incubate with shaking (110rpm) at 30?
• Max created liquid culture of Norman’s RP1 strain

REGOBricks

• Check up:
  • S. pasteurii cells did not grow in pH 9.0 Ye-Tris-NH4
  • pURE11 e. coli grew up nice and turgid
• Made autoclaved glucose solution
  • added glucose at 1g/L to S. pasteurii culture that did not grow
  • transferred new S. pasteurii to Bang media + glucose
  • transferred new S. pasteurii to Bang media - glucose
  • Plated pURE11 E. coli (S. pasteurii) on (Amp + LB) and (urea-test) plate
• Plated pURE91 E. coli (Bacillus subtilis) on (Amp + LB) and (urea-test) plate
  • question: how to plate from agar sticks
• Made glycerol stocks for pURE11, (1.4 mL in 18% v/v, stored at -80 in room 389)
• growing B. subtilis agar sticks on LB plates
• Briefly outlined mutagenesis
• Learned Norman’s way of labeling:
  • First line: What it is
  • Second line: Initials, yyyy/mm/dd Letter-# (serial number, then file down locations and specs of Letter-#)

PowerCell

• Emailed Daniel Ducat of the Silver Lab to acquire cscB S. elongatus strains
• Identified heterocyst, vegetative cell specific promoter candidates to be used in construct design
  • HetR, patB are good heterocyst candidates
  • psaC is a good vegetative candidate
• Identified sequences for above promoters, as well as glf, invA, cscB genes to be used in construct design
• Norman’s strains retained their plasmids!
• Transferred Norman’s strains into LB amp+ media,
  • Sterile technique tip- don’t drop pipette tip into media when transferring to liquid culture, end of pipette tip touches gun, is dirty

June 22, 2011

• Several equipment demos from mike on how to use the hemocytometer, microscope in the culture lab, spectrophotometer, and fluorescence meter
• First cell counting experiment; determined rough parameters for converting between OD(600nm) and cell density.
  • Counted cells via Rothschild lab protocol hemacytometers
  • Used spectrophotometer for OD
• Plated all strains
• Obtained martian simulant
• 1L SOB stock made

PowerCell

• Most of the day was vaporized dealing with the housing crisis.
• Identified sequences for other regulatory elements (varying strength RBSes, terminator). Got them from the registry.
• Designed weak, medium, and strong cscB sugar exporter constructs for both Anabaena and Nostoc

Oops, shouldn’t have incubated with shaking. Without shaking until green, then move to shaking at 110rpm. Took cyano liquid cultures off shaking platform.

• Created liquid culture of Silver’s sucrose invertase/glucose export S. elongatus strain in BG11

Created stocks for plasmid prep

June 23, 2011

PowerCell

Made BG11 stocks except Na2CO3 Made 50mg/mL streptomycin stock

Plasmid prepped Norman’s strains using Norman’s plasmid prep protocol:

• D1: pUC18@DH5a NW20110620.41
• D2: pSB1A3-P1010@DB3.1 NW20110620.A2
• D3: pSB1A10-P1010@DB3.1 NW20110620.A3
• D4: pSB1A10-J133452@DH10B NW20110620.A4

• E1: RP1@HB101 EM20110620.A1-
• E2: PSB3k3-P1010@DB3.1 EM20110620.A2
• E3: PSB3k5-P1010@DB3.1 EM20110620.A3
• E4: PSB3k5m(>-)-P1010 DB3.1 EM20110620.A4-

-During plasmid prep, 1ml of Sol2 (instead of 300ul) added; user spun down samples, removed some of Sol2, before adding Sol3; asterisked samples already had some Sol3 added before spinning and removing (therefore some of the cells may have already lysed, the DNA was poured off)

Finalized construct design (see construct page)

REGOBricks

• Impromptu precipitation expt:
  • added 10ul of liquid culture from fridge, 1M CaCl2/urea onto a microscope slide, coverslip
  • samples: E coli + pUre91, E coli + HB101, S.p. in Bang media
  • samples plated at 5pm
• OD/cell density:
  • Collected OD measurements at 10x and 100x dilution with 600 nm
  • obtained cell counts at 1x, 10x, 100x dilution (1000x+ has no visible cells)
• Grew S. pasteurii in 100 mL Bang (out of Bang media)
• Began planning directed evolution experiment in earnest

June 24, 2011

PowerCell

Transferred cyanos to 10ml cultures in square flasks created Na2CO3 stock cryostocked norman’s plasmids (see 6/23/11) Silver’s S. elongatus isn’t doing so well Learned about gibson assembly, designed primers for constructs

REGOBricks

• Julius’ impromptu precipitation experiment yielded beautiful microscope pictures

**evidence of cell crystals from both e. coli and s. pasteurii

• Made glycerol stocks of subtilis samples

• Impromptu precipitation expt:
  • made 500 mL bang media, filter sterialized
  • made 100 mL 5x bang media, to add to 400 mL 1.5% agar mix to made bang plates
• Obtained lunar simulant
• New slide protocol started at 2:55 pm.
  • Add 20µL of cell sample to top of microscope slide (amount added was unstandardized)
  • Add 10 µL equimolar Urea/ CaCL2 (1M each)
  • Drop cover slip on top of sample
  • Seal edges with nail polish
  • Observe under microscope slide
  • Incubate at 37˚ for

FRETSensor

• 71 tubes of 100uL of competent cells made from TOP10, 500 mL SOB used