IGEM:Harvard/2006/cyanobacteria/peng labbook

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Contents

Notebook

Below are notes taken during meetings or during lab; they will be either in pdf or below depending on platform used. Let me know if you need a native OneNote copy instead.

June 12th

Morning

Afternoon

  • Explained BioBricks Format
  • Created 1% Agarose Gel
  • Folding DNA nanostructures
  • Transformation of R0010, E7104, E0241 into Top-10 Competent E. Coli
  R0010 - Lac operon promoter
  E7104 - T7 promoter + GFP
  E0241 - GFP

June 13th

Morning

Afternoon

  • TF's created a 1% Agarose gel which we ran our DNA scaffold samples on
    • 130V, 45m+, imaged afterwards
 DNA Scaffold types
    -both
    -scaffold only
    -oligos only
  • Checked plating of transformant cells; no growth on control
  • Grew transformant cells in liquid culture (5mL) overnight with 50uL ampicillin
  • More brainstorming - Perry presented his domain swapping experiment, discussed DNA nanostructure box

June 14th

Morning

  • DNA Miniprep of transformant colonies
    • Out of 5mL of liquid culture, reserved 1mL for gylcerol+freeze and 4mL other
    • pelleted E. coli for each sample
    • followed QIAprep Miniprep Kit for Microcentrifuge directions
      • Used warm dH20 for elution
      • Put at 40C for ~2 min to evaporate ethanol before elution
      • Forgot to label after elution --> don't know what is what
        • Sol'n: During digest will have to run PCR, can tell R0010 from rest, but E7104/E0241 is only different by 30bp; if doesn't work can flip and try two experiments.
      • Nanodrop demonstration
Nanodrop results
  -1: 31.2ng/uL 260/280:1.75 260/230:1.92
  -2: 38.5ng/uL 260/280:1.83 260/230:1.87
  -3: 33.8ng/uL 260/280:1.70 260/230:1.44

PROBLEM: Messed up labeling of the plasmids! To diagnose, ran a 15min e-Gel to find out which is R0010.

  • Digestion of vector/insert
    • Digested R0010 (200bp cutout) as vector at S and P site.
      • .5uL Spe1, .5uL Pst1
      • 11uL h20
      • 2.5uL 10X BSA
      • 2.5uL #2 NebBuffer
      • 8uL DNA
    • Digested other 2 (~900bp cutout) as insert at X and P site.
      • .5uL Xba1, .5uL Pst1
      • 11uL h20
      • 2.5uL 10X BSA
      • 2.5uL #3 NebBuffer
      • 8uL DNA
    • Incubate @ 37C for 1h
  • Phosphatase
    • 80C@15min to kill enzyme activity
    • Used CIP (1 unit) into the R0010, 1h@37C
  • Run on 1% agarose gel
  • Image, Cutout, and Purify
    • Can isolate the three from the gel

Result

"results of PCR"
"results of PCR"
 Ladder=1kb+
 Lane 1=R0010 (#1)
 Lane 2=E0241 (#2)
 Lane 3=E7104 (#3)

June 15th

Morning

  • Brainstorming with Pam
    • Infeasibility of DNA nanostructures
    • (Linked)
  • Conduct ligation reaction
    • 6uL insert, 2uL vector, 2uL buffer, 10uL other buffer
    • 5 min incubation @ RT
  • Conduct transformation
    • 20mL cells for positive, negative, and exp. ea (top 10)

Afternoon

  • Plate transformant cells on LB-CARB
  • Brainstorming session in the afternoon
    • Linked

June 16th

Morning

  • Talk with Prof. Shih about DNA nanostructures and vailidity
    • Linked

Afternoon

June 17th (Sat)

Afternoon

  • Met up in the evening to conduct research on cyanobacteria with Hetmann, Dave, and Jeff

June 18th (Sun)

Afternoon

June 19th (Week 2)

Morning

  • Presentation of four projects
    • DNA nanostructures (Matt Katie Valerie Tiffany)
    • Fusion Proteins (Perry)
    • Universal Cell Signaling (Lewis)
    • Cyanobacteria (Peng Hetmann David Jeff)
  • Feedback on cyanobacteria
    • Found two sources for our plasmids: WHOI and MIT links
    • E. coli link may not work, but should figure out synthesis
      • Codon Devices can do it for $0.30/bp
    • Prof. Alexander van Oudenaarden works with cyanobacteria, [1]

Afternoon

  • Prof. Shih's discussion on the honeycomb lattice
  • Check plates for GFP expression
 Two colonies on experimental (R0010+E0241)!
 Many on +
 None on control
  • Grow R0010+E0241 in 5mL LB + 50uL Amp (5mg/uL orig) overnight @37
  • Further brainstorming
  • Emailed Eric Webb about Fedex #; Peter Weigele can give us PCC7942 on Thurs.

June 20th

Morning

  • Ordered culture BC11 by Sigma
  • Alain ordered PCC7942 and WH8104 strain from WHOI
    • Need to look up if other strains grow faster / better to culture
  • Need to get ahold of Prof. Knoll and vanO lab to ask about cyanobacteria culture
  • Make glycerol stocks of R0010+E0241
    • 100uL 50% glycerol, 100uL liquid media
  • DNA Miniprep
    • Follow handout; had 2 5mL samples, only used one of them
    • Tubes 0.5mL PCR; labeled in blue on top
  • Digest for diagnosis
    • Xba1 and Pst1
      1. .5uL Xba1, .5uL Pst1
      2. 11uL h20
      3. 2.5uL 10X BSA
      4. 2.5uL #3 NebBuffer
      5. 8uL DNA
      6. Incubate @ 37C for 1h

Afternoon

"Result from digest. Lane 7 is our reaction, and the ladder is 1kB+. Despite weak signal it looks as if the digest went okay; differences in size probably due to different restriction sites used."
"Result from digest. Lane 7 is our reaction, and the ladder is 1kB+. Despite weak signal it looks as if the digest went okay; differences in size probably due to different restriction sites used."

June 21st

Morning

Afternoon

June 22nd

Morning

  • Working incubator!
  • WH8102 came in the mail with SH media

Afternooon

  • Went to go pick up PCC7942 and 6803 from Peter Weigele @ MIT Building 68
  • Made liquid culture without thiosulfate
  • Made 6 samples
    • PCC7942 streak w/toothpick
    • PCC7942 single w/toothpick
    • PCC7942 single w/o toothpick
    • PCC6803 streak w/toothpick
    • PCC6803 single w/toothpick
    • Control w/ toothpick
  • Sprayed with EtOH beforehand working area
  • 16h day / 8h night, shaking, open lid, 30C

June 23rd

Morning

  • Measured light intensity with lux meter
    • Turns out only a little region has ~4200 lux; rearranged samples
  • Tested plastic cover; does not diffuse light

Afternoon

  • General research on ideal conditions for growth

June 26th (Week 3)

Morning

  • Morning meetings
  • Made more BG11 liquid media w/ thiosulfate
  • Made 500x Thiosulfate+Na stock sol'n
  • Brought down solid plate recipe

Afternoon

  • Checked experiments
    • See Jeff's pictures here
    • See results for the day here
  • Created new liquid colonies
    • 125mL BG11 liquid in autoclaved glass 500mL earlemeyer
    • PCC7942 dried out!
  • Question to be addressed:
    • How do the plasmids work? DNA delivery?

Evening

  • Emailed Prof. Susan Golden

June 27th

Morning

  • PCR tutorial
    • Tm = 65, y = Tm -5
    • deltaG > -3 kCal/mol

Afternoon

  • Beginning primer design
  • Ordered PCC7942 from atcc.org

June 28th

Morning

  • Sick :(

Afternoon

  • Reviewed information from Prof. Golden
  • Growth in PCC6803!
  • Completing primer design for KaiABC extraction

July 6th

Morning

  • Rehydrated oligos that arrived (10 of them)
    • First in 250uL dh20
    • Then diluted to 20uM ea
  • PCR (colony) from dried PCC7942 plate
    • Did not work
  • Decided to model oscillation

July 7th

  • Recieved PCC7942
  • PCRed again, this time with liquid culture as template
  • 2 liquid cultures made, 500mL flask w/100 mL
    • 1 w/ thiosulfate
    • 1 w/o thiosulfate
  • 2 solid cultures made
  • Modeling

Post July 7th

Look into our cyanobacteria page at IGEM:Harvard/2006/Cyanobacteria/Notebook.

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