Team:Brown-Stanford/Lab/Notebook/Week6

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Jamboree

July 18, 2011

imaged PCR: RBSm|cscB on 7/15, according to 7/11 parameters (may need to remove from thermocycler):
first gel from 7/18: unable to see ladder or bands (restained with fresh GelStar, then recast a fresh gel to try loading again)
did not use TAE for post-stain; HAVE TO USE TAE + GELSTAR FOR POST STAIN OR ELSE DNA WILL DIFFUSE OUT OF GEL
gel extract and/or PCR cleanup to prepare for Gibson
imaged PCR: second round Ana wk/med/str, Nostoc wk (from 7/15)
Gel extracted and PCR cleanup directly on rbs-GFP-TTsp (bands cut 7/15, PCR product from 7/12); Nanodrop results: PCR cleanup-120ng/ul, Gel Extract- 19ng/ul
possibly run a 2nd round PCR? (don’t think so)
PCR (put in at 6:30pm):
2nd round PCR on RBSw cscB, RBSs cscB, cscB TTsp from 7/15 gel exts
redid second round PCR on Ana wk/med/str, Nostoc wk, RBSm|cscB, (+) control 15B
Jovian and Lei tested sucrose assay

Finally got the constructs ordered (3 from Invitrogen, and the cohesin-cohesin from DNA 2.0)

imaged PCR from 7/18 (9 samples); MAKE SURE TO STAIN WITH 5ul GelStar in 50ml TAE
gel bands visible/cut/extracted for RBSw cscB (~10ng/ul), RBSm cscB (~5ng/ul), RBSs cscB (13.9ng/ul),
Jovian extracted bands for Ana med, Nos weak: Nanodrop readings in the negatives? REDO
processed ATCC E coli 9637 into liquid culture
resuspended dried cells into 450ul LB, put 150ul of suspension with 2ml of LB, placed in 37C at 3pm (repeated 3x)
set up an 85ml BG11 N+ culture for Anabaena 7120 and N. punctiforme (75ml BG11N+ and 10ml of the existing saturated liquid culture)

Consolidated protocols for transformations, including:
- Soil
- Protoplast
- Protoplast electroporation
- Standard

PCRed: the never-successful ones: cscB|RBSgfp; Nostoc strong RBS; Ana weak/str (did not see bands)
made 3x cryostocks of E. coli W (slots 47, 48, 49 in PowerCell -80C box); used 20% glycerol
imaged the PCR from this morning (7/20):
under 2% agarose, poststaining for 30min is inadequate; Kosuke says to use fresh GelStar in polyethylene container, stain for more like 45min
INSTEAD, begin to cast gels with GelStar embedded, use 6ul of ladder
second gel (Gel #1b) cast, loaded with all 15ul of remaining PCR product; 6ul of ladder
Anabaena strong showed strong bands, bands cut, ready for extraction
Lei transformed 2x cscB plasmid into TOP10
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
Sigma order was placed with Mike (b-NAD purchased, 600mg)

sick! :(

Began preparing for the arrival of pUB110
- Made solutions for 4 transformation protocols
- Autoclaved potting soil in preparation for soil transformation

- Performed OD curve with 1x, 1/2x, 1/5x, 1/10x, and 1/100x with Bang media and cuvettes. Instead of plating, used haemocytometer to count cells in all samples.Came up with a final cell count equation, Cell count/mL = (OD - .0746)x10E9

7-23

Began dried filter paper experiment:
[Sterile in UV] Took nitrocellulose paper**, put into on large plastic petri dishes
12 sheets of paper, divided into 2 x [control, 10x dilution, 100x dilution, 1000x dilution and 10000x dilution] of S. pasteurii (OD600: 1._____)
prewet nitrocellulose paper with 100 ul of DI water, tip plate around to spread it out
put 50 ul of each cell dilution onto each paper, spread evenly
incubate paper in giant petri dish in 30 C

Controls II: repeat same procedure, but instead of incubating the paper, immediately flip the paper (cell containing side down) onto Bang media plates. Incubate plates at 30 C

Grew up
- 2x 2mL B. subtilis in LB media
- 2x 2mL E. coli in LB media + amp
  - caution, nitrocellulose paper is extremely flammable

gel extraction on Ana strong from 7/20 PCR; Nanodrop 7.2ng/ul
imaged Nostoc strong (from 7/20 PCR); moderately strong bands cut, extracted (Nanodrop 5.5ng/ul)
made 1L BG11 N+ from 50X stock
PCR: Phusion PCR
tried gradient PCR 40-50C using phusion polymerase; samples Ana mid, Nos mid, RBSm|cscB, pos cont (well 15B)
used 1.5ul of template in 20ul rxn; added 3% DMSO
times: initial colony popping with 98C for 5’; initial denature 30sec @ 98C; 10sec @ 98; 20sec @ 40-50C; 15sec @ 72C; repeat 32x; final elongation 10min @ 72C
Phusion is FAST, yo
pick colony of CscB transformant, start 2ml LB amp+ liquid culture (three replicates); placed in 37C at 7:15pm

Imaged Phusion PCR from 7/22; 4x bright visible bands cut from all samples (Ana med, Nos med, RBSm|cscB)
gel extraction on bands (ProMega kit; re-eluted samples through column three times on first and last steps in order to capture maximum DNA)
Nanodrop readings: RBSm|cscB 40.1ng/ul; Ana med 21.1ng/ul; Nos med 14.2 ng/ul
Started more 100ml cultures of Anabaena and Nostoc; BG11 N+, 2x of each
Plasmid prep of liquid cscB cultures
plasmids in 4C of Rm335, “Norman’s Plasmids” rack; Nanodrop readings of 100.6, 79.4, 92.2ng/ul
Dry autoclave run for tips and glassware (with Biocementation team)
embed bacteria on nitrocellulose paper for monday balloon flight