IGEM:Harvard/2006/cyanobacteria/peng labbook

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

• Notes from first meeting (pdf)

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

• Brainstorming by presenting previous projects
• Past_project_notes

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"

 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

• Talks on two papers
  • iGEM:Harvard/2006/Brainstorming_Papers_-_Zhipeng
• Other talks can be found at IGEM:Harvard/2006/Brainstorming

June 17th (Sat)

Afternoon

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

June 18th (Sun)

Afternoon

• Finalized information and created Powerpoint
  • IGEM:Harvard/2006/Cyanobacteria for link to cyanobacteria information
  • Media: Presentation.ppt for Powerpoint delievered (requires Beta 2007 to work correctly)

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

• Research into cyanobacteria: see IGEM:Harvard/2006/Cyanobacteria for contribution
• Digest gel result

"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

• Populating IGEM:Harvard/2006/Cyanobacteria
• Organizing shopping list/calling stores

Afternoon

• Roadtrip to Home Depot to obtain items
• Wrote Guide to building a cyanobacteria incubator
• Built incubator
• Will pick up PCC7942 tomorrow.

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.