Team:Brown-Stanford/Lab/Notebook/Week7

• 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 24, 2011

REGOBricks

• Control II of yesterday's experiment grew colonies in 10x and 100x dilution, colonies located at edges of nitro paper

• Attempted revival of ? of yesterday’s filter paper experiment:
  • flip the paper upside down (side containing cells facing down) onto new Bang plates. The moisture of the plate covers the paper.
  • On one half of the paper, add 50 ul Bang media, to wet/wash the cells onto the plate.

• Repeat dried filter paper experiment with Subtilis (OD 0.996) and E. coli (OD 1.12) grown up from yesterday.

• Transformation of E. coli (competent Top10) with D1 plasmid containing amp resistance

• Growing up 50 mL of E. coli containing cscB plasmid in LB + amp

• Made BANG plates with a gradient of Kan to test for S. pasteurii survival
• Data will be used to make plates to select for Kan resistance in transformed pasteurii

July 25, 2011

PowerCell

• Perform additional Phusion PCRs:
• Round 1: cscB|TTsp; cscB|RBSgfp; rbsGFPttsp; (no pos control)
• cscB|TTsp bands visualized, cut; the rest were inconclusive; redo
• cscB|RBSgfp was distorted on gel; both CG and GFP exhibited lots of secondary amplifications; try higher specific
• make BG11 N+ plates (in prep for NDA) (see NDA3 design document)
• Test transformation - liquid cultures of conjugative transformation E. coli lines
• pRL1383a - from plate
• pRL443 - from plate
• RP1 - from cryo
• pRL25 - from plate
• pDS4101 - from agar
• make liquid culture of E. coli W for NDA3 - from cryo
• Innoculated 5 more 500mL liquid cultures for both Ana and Nos
• Tranformed J45120 and J45200 for long (+) conts. for PCRs

FRETSensor

• Started planning directed evolution experiment for UV resistence
• Ordered protein purification columns

July 26, 2011

PowerCell

• gel extraction on cscB|TTsp from 7/25/11
• PCR cscB|RBSgfp; rbsGFPttsp (WITH PROPER TEMPLATE); Ana str, Nos str, RBSs|cscB; gradient from 55-45C
• design primer for prom-GFP-ttsp construct;
• Prepare test transformation with prepared liquid cultures (eek)
• make three classes of plates (BG11 agar + 5% LB; BG11 agar + spec; BG11 agar + kan)
• BG11 + LB agar plates (more like ~160ml BG11, 16.6ml LB, ~180ml agar); 133ml BG11 + ~133ml agar + 335ul spec100; 133ml BG11 + ~133ml agar + 335ul kan20
• See cyanobacterial transformation document for protocol
• Liquid cultures of transformation plasmid strains, again.
• transformed j45200 long pos cont.
• gel extracted cscb|TTSP phusion PCR. 32.9 ng\ul
• miniprep long pos controls
• PCR m.AvaI??????????

FRETSensor

• Exposed e coli in LB to 10s, 30s, 60s, 90s, 120s, and 180s, UV in falcon tubes.

July 27, 2011

PowerCell

• Image PCR from 7/26 (rbsGFPttsp; cscB|rbsGFP; Ana str; Nos str; RBSsCscB);
• samples are still in the gradient cycler in Innovation Lab (turn off after removing tubes!);
• run the 20 lanes from 7/26 (five templates x four temperatures), re-run the four lanes of Nostoc med part (from 7/21 PCR) in order to obtain more gel extracted product
• Gel 1 (0.7%): 1kb ladder, rbsGFPttsp; cscB|rbsGFP
• Gel 2 (2%): 100bp ladder, Ana str, Nos str
• Gel 3 (2%): 100bp ladder, RBSsCscB, Nos med
• Try to doublecheck GelRed procedure with Thomas before making gels
• Extract any good bands (follow Thomas’ suggested modifications to maximize yield), Nanodrop readings
• Another stab at transformation!
• made BG11+1% Agar+5% LB, BG11+1% Agar+Spec, BG11+1%Agar+Kan plates
• Continued transformation begun 7/26 with Julius’ plates, incubating for one day in 30?
• Began new transformation on new plates, incubating in 30?
• Setup cross of pDS4101 and pRL25 to incubate overnight, to be streaked tomorrow on amp/kan LB.
• Liquid cultures involved in conjugative transformation are stored in 335 4?.
• Ran Gels on Julius’ PCR from 26Jy2011 (Cscb|RBSgfp, RBS-GFP-TTSP, RBSs|cscB, Ana S, Nos S) cut out bands.
• Lots of extraneous amplification, especially in cscB|RBSgfp - cut out bands in right places, but sort of dubious they’re the right things...
• Innoculated 4mL liquid stock of long pos cont. for mini prep.

FRETSensor

• All of LB/Falcon tubes grew at all dilutions
• Next, we exposed in open LB (2 mL)

July 28, 2011

PowerCell

• Streak pDS4101xpRL25 onto amp/kan (lei/jovian)
• gel extraction of 1st round PCR’s
• miniprep of long positive control from registry
• 2nd round PCR’s
• Gel of 2nd round PCR’s
• Gel extraction of 2nd round PCR’s
• continue transformation-post-conjugation cells streaked onto antibiotic BG11 for attempts 1 and 2
• lab cleaning at 1:30

REGOBricks

• Successful balloon launch this morning (Max tell us more!)
• Plasmid prepped pUB110 using the modified qiagen protocol w/ lysozyme.
• Ran a gel to confirm the presence of the plasmid which is ~4500 bp
• Today was lab clean up day. Yipee

FRETSensor

• All LB grew again! :(

July 29, 2011

PowerCell

• ran gel of 2nd round PCRs
• cut out bands, gel purified
• successfully got
• PCR amplification of pSB1C3 and pSB1A3 with g1001, g0001 primers
• Create and aliquot Gibson master mix

July 31, 2011

REGOBricks

• Preparation of frozen S. pasteurii aliquot:
• Centrifuge stock of 25 mL S. pasteurii grown up, OD 1.377
• using Andre’s OD/cell count curve,
  • original procedure called for 200 mL of OD 0.2 (total cell count of ______)
  • current procedure uses 25 mL of OD 1.37 (total cell count of ______)

FRETSensor

• Planned next directed evolution experiment
• Made more LB liquid