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
 * [[Media:ZS_brainstorm.pdf | 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 DNA Scaffold types -both -scaffold only -oligos only
 * TF's created a 1% Agarose gel which we ran our DNA scaffold samples on
 * 130V, 45m+, imaged afterwards
 * 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 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
 * 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

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

Ladder=1kb+ Lane 1=R0010 (#1) Lane 2=E0241 (#2) Lane 3=E7104 (#3)

June 15th
Morning Afternoon
 * 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)
 * Plate transformant cells on LB-CARB
 * Brainstorming session in the afternoon
 * Linked

June 16th
Morning Afternoon
 * Talk with Prof. Shih about DNA nanostructures and vailidity
 * Linked
 * 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 Afternoon Two colonies on experimental (R0010+E0241)! Many on + None on control
 * 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,
 * Prof. Shih's discussion on the honeycomb lattice
 * Check plates for GFP expression
 * 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 Afternoon
 * 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
 * .5uL Xba1, .5uL Pst1
 * 11uL h20
 * 2.5uL 10X BSA
 * 2.5uL #3 NebBuffer
 * 8uL DNA
 * Incubate @ 37C for 1h
 * Research into cyanobacteria: see IGEM:Harvard/2006/Cyanobacteria for contribution
 * Digest gel result

June 21st
Morning Afternoon
 * Populating IGEM:Harvard/2006/Cyanobacteria
 * Organizing shopping list/calling stores
 * Roadtrip to Home Depot to obtain items
 * Wrote Guide to building a cyanobacteria incubator
 * Built incubator
 * Will pick up PCC7942 tomorrow.

June 22nd
Morning Afternooon
 * Working incubator!
 * WH8102 came in the mail with SH media
 * 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 Afternoon
 * Measured light intensity with lux meter
 * Turns out only a little region has ~4200 lux; rearranged samples
 * Tested plastic cover; does not diffuse light
 * General research on ideal conditions for growth

June 26th (Week 3)
Morning Afternoon Evening
 * Morning meetings
 * Made more BG11 liquid media w/ thiosulfate
 * Made 500x Thiosulfate+Na stock sol'n
 * Brought down solid plate recipe
 * 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?
 * Emailed Prof. Susan Golden

June 27th
Morning Afternoon
 * PCR tutorial
 * Tm = 65, y = Tm -5
 * deltaG > -3 kCal/mol
 * Beginning primer design
 * Ordered PCC7942 from atcc.org

June 28th
Morning Afternoon
 * Sick :(
 * 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.