Team:Stanford/Notebook/Lab Work/Week 1

6/28 Monday

General Meeting Notes

• Wiki stuff
  • Everyone should sign up on 2010.igem.org website
    • Take advantage of the new calendar system
    • Need to transfer current website over
  • Current wiki needs a new vision
    • Francisco + Alex assembling wiki committee
• Transform the first wave of cells
  • Pick colonies
  • Colony PCR
  • Sequence parts to validate
    • Use the Smolke sequencing network
• Characterize promoters
  • Digest, ligate
  • Need to acquire enzymes
  • Need an PO account.
    • Email Jeanne
• Get competent cells
• Getting DNA
  • Assembly PCR
  • PCR from host cells
  • Synthesize de novo
• RNA team
  • Use RNAstructure
    • Maybe use ViennaRNA or dinamelt
  • Talk to Arkin and Berkeley Postdocs
  • Finalize sRNA library
  • 9kb plasmid
• Meeting scheudle
  • Monday morning is good for logistics
  • Group meetings for presentations
    • One person presents their work in the context of everything else
  • Journal club
  • Karina will set up a doodle

Alex's Notebook

• Transformations using DH5alpha cells from Graham (also have TOP10)

• Protocol:
  • 1) Take out competent E.coli cells from –80 C freezer.
  • 2) Turn on water bath to 42 C.
  • 3) Put 50 ul of competent cells in a 1.5 ml tube (Eppendorf or similar).
  • 4) KEEP EVERYTHING ON ICE UNLESS OTHERWISE STATED.
  • 5) Add 50 ng or 2.5/5 ul of circular DNA into E.coli cells. DO NOT PIPETTE UP OR DOWN; STIR INSTEAD! Incubate on ice for 15/30 min.
  • 6) Put tube(s) with DNA and E.coli into water bath at 42 C for 20 seconds.
  • 7) Put tubes back on ice for 15 minutes to reduce damage to the E.coli cells.
  • 8) Add 1 ml of SOC. Incubate tubes for 1/2 hour(s) at 37 C.
  • 9) Spread about 100/200 ul of the resulting culture on LB plates (with the appropriate antibiotic added – usually Ampicillin or Kanamycin). Grow overnight.
  • 10) Pick the colonies about 12-16 hours later.

• Parts:
  • Plasmids:
    • - 1A2
      • P1, 11P, BBa_J04450. 100-300, pMB1, 2079.
    • - 1A3
      • P1, 1C, Ba_J04450. 100-300, pMB1, 2157.
    • - 4A5
      • P1, 1G, BBa_J04450. ~5, pSC101, 3395.
    • - 3C5
      • P1, 3C, BBa_J04450. 10-12, p15A, 2738.
    • - 2K3
      • P1, 5C, Insert: BBa_J04450, star. Length: 4425 bp.

• • Promoters:
    • - Bad (BBa_I0500). Input(s): L-arabinose. Output(s): PoPS. Need with mutated strain. Tested range: 2e-4% up to 2e-1%. Can probably go lower. (http://mic.sgmjournals.org/cgi/reprint/147/12/3241).
      • P1, 14N, Plasmid: pSB2K3. Length: 1210 bp.
    • - TetR and LuxR (BBa_F2620). Input(s): C10HSL. Output(s): PoPS. Tested range: ~1e-7 up to 1e-4. Can probably go to a higher concentration. (http://partsregistry.org/wiki/images/f/fe/EndyFig1-F2620DataSheet.pdf)
      • P2, 6E, Plasmid: pSB1A2, star. Length: 1061 bp.

• • Reporter:
    • - Endy’s GFP (BBa_E0240).
      • P1, 12M Plasmid: pSB1A2, star. Length: 876 bp.

• • Miscellaneous Parts:
    • - GFP Producer Controlled by 3OC6HSL Receiver Device (BBa_T9002). For use with C10HSL (homoserine lactone; http://www.sigmaaldrich.com/catalog/ProductDetail.do?lang=en&N4=17248|SIGMA&N5=SEARCH_CONCAT_PNO|BRAND_KEY&F=SPEC)
      • P2, 9A, Plasmid: pSB1A3, star. Length: 1945 bp.

6/29 Tuesday

Alex's Notebook

• LB agar plates

• Recipe (per 500 ml bottle):
  • 5 g Bacto-Peptone
  • 2.5 g Yeast Extract
  • 2.5 g NaCl
  • 6 g Agar

• From recipe, measure bacto-peptone, yeast, NaCl, and agar into each bottle. Add 450ml of water from the millipore filter in the washroom and add a stir bar. Mix on the stirplate. Remove stirbar with stirbar retriever and pour most of the mixture into a graduated cylinder. As you get to the bottom of the bottle, swirl the mixture and pour. Bring up to final volume of 500ml and pour back into the original bottle. Swirl and pour as you transfer to make sure as much of the mixture is returned to the bottle.
• Adhere autoclave tape. Loosely cap bottle and set in autoclave tray with 2 inches of water. Just prior to closing the door, give each bottle another swirl to ensure mixture has not settled out.
• Autoclave on Liquid 60.
• Individually remove each bottle and gently swirl contents. Let cool until it can be held in your hand for a minute, add antibiotics, and pour into plates.

• A: 50 ug/ml
• K: 20 ug/ml
• C: 34 ug/ml
• T: 20 ug/ml

• Links:
  • http://openwetware.org/wiki/Preparing_chemically_competent_cells
  • http://openwetware.org/wiki/Endy:Pouring_LB_plates_from_prepped_media
  • http://openwetware.org/wiki/Designing_primers
  • http://openwetware.org/wiki/Smolke_Endy_LB_Agar

Laura's Notebook

• made LB agar (with Alex and Chris)
  • made 1 500 mL bottle, 3 250 mL bottles, then autoclaved

6/30 Wednesday

Laura's Notebook

• Re-try failed transformation (failed 2X for Alex and Chris)
• Protocol followed:

1. get competent cells from -80oC freezer
2. set H2O bath to 42oC (already done)
3. add 50 uL cells to a 1.5 mL tube (keep tubes on ice)
4. add 50 ng or 2.5 uL circular DNA to E. Coli (variation: used 5.0 uL I0500 this time)
5. on ice 30 min
6. 24oC H2O bath 20 sec.
7. on ice 15 min
8. add 1 mL SOC, 2 hours at 37oC (rotating)
9. plate100uL on appropriate plate (kanamycin this time), incubate 12-16 hours before picking colonies

• variations: 200 uL plated this time, also plated on kan/X-gal/IPTG plate (plasmid inducible copy # by X-gal)

7/1 Thursday

Laura's Notebook

• did 8 minipreps from Chris'/Alex's cultures
• used Qiagen spin kit, with the following variations:

1. skipped "recommended" wash step
2. eluted with H2O

• Concentrations from Nanodrop (taken by Chris B.)

• helped Karina, Francisco with the transformations of parts

Francisco's Notebook

• Learned how to transform using electroporation.

7/2 Friday

Recap Meeting Notes

• Things to think about:
  • name of the device (see if electronic analogue exists)
  • decouple the small RNA binding from the target gene ("RSID" tags)
  • leadership structure for the team (weekly leader order on Google Calendar)
  • analog sensor- redundancy at device level or component level?
  • July 16: presentation to MDV- potential donor of $

Francisco's Notebook

• Miniprepped Parts. Nanodrop results below: