Team:Brown-Stanford/Lab/Notebook/Week7

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Jamboree

July 24, 2011

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

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

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

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??????????

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.

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

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 ______)