Harvard iGEM 2005 Schedule

From FreeBio

Contents

To Do

Week 3 and on

Every Monday, 10am, the whole team meets in Biolabs 1058 and presents progress

  • More brainstorming
  • Test already characterized devices
  • Start ordering DNA
  • Assemble and test new parts

The BioWire schedule outline is on the main page. Specific experiments are scheduled on the Materials and Methods page.

Other

Every Wednesday, 4pm, ALife Boston seminar in Biolabs 1058 is open to the public

Jamboree

5th & 6th of November, 2005

  • all schools will meet to discuss their results.

Completed

Preliminaries

Kick-off meeting

5:35 to 7:30 PM Thursday May 12, at 16 Divinity Ave, Biolabs 1058.

  • Introductions - team members met each other and all of the faculty members.
  • Discussed initial ideas about the project.

Teachers' workshop

MIT Room 32-144, Stata Center, May 14-15, 2005

  • summarize the project on the main WIKI (throughout the summer)
  • make parts freely available to all
  • communicate!

June

June 1 (Wed)

10:00-10:30 (Pam, Biolabs 1058)

  • Introductions
  • Brief general history and overview of synthetic biology
  • What do we want to accomplish this summer

10:30-12:30 (Kit, Biolabs 1058)

  • How to be organized in science
  • Brainstorming strategies
  • Establish milestones
    • Jamboree - Nov 5/6
    • Classes start - Sep 17
    • Science done, formatted, saved - Sep 1

12:30-2:30 Lab Safety Training (room 177, Fairchild, 7 Divinity Avenue)

  • Lunch Provided.

2:30-6:00 Introduction to the Lab (Teaching Lab)

  • PCR
    • Paula, Yves, Sasha

June 2 (Thur)

10:00-10:45 (George, Biolabs Main Lecture Hall)

  • Overview of synthetic biology -- Slides [1]

10:45-11:30 (Radhika, Biolabs Main Lecture Hall)

  • Overview of last year's projects

11:30-1:30 Brainstorming (Biolabs 1075)

  • Lunch Provided.
  • Discussed possible projects
  • Discussed possible team names
Initial Ideas

1:30-4:30 Introduction to the Lab (Teaching Lab)

June 3 (Fri)

10:00-11:00 (Kit, Biolabs 1058)

  • What is a machine?

11:00-10:30 (Radhika, Biolabs 1058)

  • Modelling
    • ODEs
    • Cell circuits

12:00-1:00 Brainstorming (Biolabs 1058)

  • Lunch Provided.
  • Ran project ideas by faculty
    • All sound good, including Etch-a-Sketch, except password is probably too complex.

1:00- Introduction to the Lab (Teaching Lab)

1:15-2:30 Introduction to MatLab (Biolabs 1058)

2:30- Scheduling and Group meetings (Teaching Lab)

  • Over the weekend, each group will do:
    • Literature and background search
    • Determine steps required
    • Determine input and output
    • Risk analysis
    • Meet to prepare presentation
  • Present on Monday to entire team
  • Don't forget Logo, team-name, t-shirt design -- ASW

June 6 (Mon)

Please log in (top right) while you make edits.

9:30-10:45 (George, Biolabs 1058)

10:45-12:45 (Biolabs 1058)

  • Discussion of project ideas

1:00-3:15 (Randy, Biolabs 1058)

3:15-5:00 (Teaching Lab)

  • Redo of transformation
    • Plates from last time were bad: all plates showed growth (AMP non-functional)
    • Other plates in same batch had liquified
    • Had fresh plates made

June 7 (Tue)

10:00-10:45 (Sasha and Kang-Xing, Biolabs 1058)

  • Information, Abstraction, and Digital Logic -- Slides (PPT)

10:45-11:20 (Yin, Biolabs 1058)

11:20-11:25 (Yin, Biolabs 1058)

June 8 (Wed)

10:00-12:00 - Discuss projects some more

16:00-17:00 - Julie Norville on Building protein crystals by engineering methods.

June 9 (Thur)

10:00-1:15 - BioWire group met in 1058. Check out the redesigned wiki pages.

June 10 (Fri)

10:00 - BioWire group meeting in 1058

10:00 - BioSketch meeting 5th flr break room

  • More brainstorming

June 13 (Mon)

10:00-11:15am - BioSketch Group Meeting Presentation in 1058 ppt and pdf
11:15am-12:15pm - BioWire Group Meeting Presentation in 1058 Image:BioWire Progress Report, Week One.ppt and Image:BioWire Progress Report, Week One.pdf

12:15-1:00pm - Lunch!

1:00-5:00pm - BioWire Meeting in 1058
1:00-5:00pm - BioSketch Meeting in 5th flr break rm; planning experiments

June 14 (Tue)

BioSketch group:

  • 12:00-2:00pm - Planning Switch Design based on constructs of Collins group
  • 2:00-4:30pm - Planning Switch Designs based on BioBrick constructs

BioWire group:

June 15 (Wed)

BioSketch group:

  • Collins team:
    • Received all 6 plasmids from Collins
    • Received seqs for pSTMb1 & pCIE; annotated them
    • Ordered tet, chl antibiotics
    • Ordered mut primers for LacIts(241/265)

4pm Aneil Mallavarapu on Little b, a language for building modular models of biology.

June 16 (Thur)

BioSketch group:

  • Made tet, chl, kana stocks and selective plates
  • Collins team:
    • Reconstituted Collins plasmids in ddH2O and transformed them into E. coli
    • Ordered seq primers for pTSMb1 insert
    • Received remaining 4 sequences for Collins plamids; annotated them
  • BioBricks team:

June 17 (Fri)

BioSketch group:

  • Collins team:
    • Transformed pTSMb1 & pTSMb2 into E. coli
  • BioBricks team:
    • Received BB parts

June 18 (Sat)

BioSketch group:

  • Collins team:
    • Inoculated Collins plasmids for minis
  • BioBricks team:
    • Streaked BB parts onto selective plates

BioWire group:

  • Streaked BB parts onto selective plates, put into incubator at 37C overnight.
  • Reselected for single colonies for the failed minipreps of BB parts received last week, transferred colonies to culture tubes, and put tubes in 37C shaker overnight.

June 19 (Sun)

BioSketch group:

  • CollinsMod team
    • Miniprepped Collins plasmids
    • Overnight diagnostic digests of miniprep plasmids:
      • HindIII digests of pTS(pTSMa), pTSMb1, pTSMb2, pWCI(pCIE)
      • RsaI digests of pWG(pCIRa), pCIRb
  • BioBricks team
    • Inoculated for minipreps of BB parts

BioWire group:

  • Moved BB culture tubes from shaker to 4C fridge (will be miniprepped with the other BB parts on Monday).
  • Selected for single colonies on BB plates, transferred colonies to culture tubes, and put tubes in 37C shaker overnight.

June 20 (Mon)

Group meetings:

  • BioWire group:
  • BioSketch group: ppt

BioSketch group

  • CollinsMod team
    • Ran diagnostic digests, repeated twice. DNA conc too low: Overnight digest w. 5µl miniprep DNA.
    • Site-directed mutagenesis w. pTS, C0012 (for BB team), Q04121 (for BB team) using primers for Ala214->Thr (P1/2) and Gly265->Asp (P3/4):
      • Mistake in use of centrifuge/PCR tube: lost rxns for ctrl, C0012 (P1/2), C0012 (P3/4), Q04121 (P3/4)
      • pTS (P1/2), pTS (P3/4), Q04121 (P1/2) rxns were transformed and plated.
    • Ordered Sph I
    • Repeated minipreps for pTS, pWG, and pWCI w. 10ml inoculant each
  • BioBricks team
    • Miniprepped all parts

BioWire group

  • Miniprepped 28 BioBricks, performed analytical digests. 26/28 appeared to be ok, 2/28 did not show up. Both failed digests were for parts from the Las system (not immediately needed).
  • Digested 20 BioBricks for ligation tomorrow.
  • Ordered more BioBrick parts for the Las system and for aiiA.

June 21 (Tue)

BioSketch group:

  • CollinsMod team
    • Miniprepped Collins constructs, with 10ml culture.
    • Digested constructs and ran gel: Good run.
    • Growth for pTSts241 and pTSts265 transformants (QuikChange): picked colonies for 5ml inoculations.

June 22 (Wed)

BioSketch group:

  • CollinsMod team
    • Miniprepped pTSts241 & pTSts265 transformants.
    • Diagnostic digests on mutagenesis products: pTS265 minis look good.
    • Overnight single digests w. HindIII (double-cutter) and SpeI (single-cutter)

4pm Farren Isaacs on Multiplex DNA synthesis and some applications.

June 23 (Thur)

BioSketch group:

  • CollinsMod team
    • Ran gel on overnight HindII and SpeI digests: all pTS241 minis look good.
    • Cotransformation of competent cells w. pTS/pWG; also did pTS as +ve.
    • Designing cloning strategies for pEL and pWC.
    • Overnight digestion of pTS w. SphI/XbaI to make pEL.
    • Sent PCR primers for making pWC.
    • Sent off pTS241 and pTS265 samples for sequencing.


June 24 (Fri)

BioSketch group:

  • CollinsMod team
    • Co-transformation of competent cells w. pTS/pWG indicated substantial colony growth.
      • Ctrl for plates: streaking w. pTS alone and pWG alone, along w. pTS/pWG.
    • SphI/XbaI digest of pTS from previous day did not work: only linearized.
      • Ctrl overnight single-digests w. SphI and XbaI, along with the SphI/XbaI digest.

June 25 (Sat)

BioSketch group:

  • CollinsMod team
    • Testing circuits
      • Kan+Amp plates are good: no growth for pTS and pWG individually.
      • Co-transformation w. pWCI/pWG.
    • Received lacI(-) strain from BioBricks.
      • Inoculated 2ml for frozen strain collection.
      • Streaked onto fresh LB plate.
      • Looked into protocol for making cells competent: need PEG 3350 and DMSO.
    • Cloning pEL
      • XbaI does not cut at site in pTS, probably due to methylation.
      • Alternative strategy: double-digests w. pTS and pWCI to replace P(trc)-cI in pWCI w. P(L*)-lacI from pTS.
      • Overnight digests of pTS and pWCI w. BamHI/SphI.

June 26 (Sun)

BioSketch group:

  • CollinsMod team
    • Testing circuits
      • Inoculated 5ml each of pWG; pWG/pWCI; pWG/pTS
    • Preparation of lacI(-) strain
      • Prepared frozen stocks
      • Inoculated for making competent
    • Cloning pEL
      • Presumed pWCI cuts like pTS w. BamHI/SphI: wrong plasmid?

June 27 (Mon)

BioSketch group:

  • Group meeting presentation: ppt
  • CollinsMod team
    • Testing circuits
      • Miniprepped pWG/pTS and pWG/"pWCI"
      • Analytical digest w. SpeI shows that both plasmids are present
      • 5ml inoculation overnight of pWG; pWG/pWCI in TOP10 strain
    • Preparation of lacI(-) strain
      • Made cells competent: lots in -80 freezer
      • Ctrl transformation w. +ve plasmid from Invitrogen
      • Plated 100µl, 250µl, and 500µl from 1ml culture
    • Cloning pELc
      1. Digested pTS w. BamHI/SphI
      2. Klenow rxn on digest
      3. Gel purifed 1.7kb lacI insert and 4kb vector
      4. CIP on 4kb vector
      5. Column purifed of CIPped vector
      6. Ligated lacI insert into vector
      7. Transformed
    • Cloning pWCIb
      1. Digested pTS w. PstI/SphI
      2. Klenow rxn on digest
      3. Gel purified 4.8kb vector w. cI
      4. Recircularized vector w.o. lacI
      5. Transformed

June 28 (Tue)

BioSketch group

  • CollinsMod team
    • Cloning pWCIb
      • Ligation worked
      • Inoculated 5ml cultures for five colonies for miniprepping
    • Cloning pELc
      • No colony; will try to XbaI digest in dam- background.
    • Cloning pELa, pEL241, pEL265
      • Transformed pTS, pTS241(11), pTS265(1) into dam- strain
    • Cloning pTS241 and pTS265
      • Inoculated 10ml cultures for pTS241(11), pTS241(12), pTS265(1), pTS265(9)
    • Discovering the identity of pWCI (aka pCIE)
      • Inoculated 5ml culture for colony transformed from original Collins pCIE for miniprep
    • Preparing lacI- strain
      • Plates from yesterday were either confluent, or had no growth.
      1. Added 5µl pWG to 50µl competent cells
      2. Incubated 10' on ice; heat-shocked @ 42°C for 2'.
      3. Added 500ml LB. Incubated for 1h @ 37°C on shaker.
      4. Plated 10 and 50µl on LB-tet plates.
    • Microscopy
      1. To 5ml medium, add 100µl cells from culture grown overnight.
      2. Wait until OD600 = ~0.3.
      3. Spin down 1ml of cells in microfuge.
      4. Resuspend pellet in 100µl medium.
      5. Pipet 5µl onto slide.
      • The pWG alone was bright green regardless of IIPTG (0 or 2mM)
      • pWG+pTS exhibited dim fluorescence in all cells, but strong fluorescence only in select cells; again regardless of IPTG.
      • Grow overnight in IPTG.

June 29 (Wed)

4pm Joao Magalhaes on Molecular mechanisms of aging.

BioSketch CollinsMod group

  • Cloning pWCIb
    • Miniprepped five pWCIb clones
    • Analytical digests w. HindIII/SpeI: all five show expected 2 bands
  • Getting to the bottom of Collins pCIE (pWCIa)
    • Analytical digest w. HindIII/SpeI shows identical banding to pTS
  • Reconstructing Collins circuit
    • Co-transformation of pTS/pWG, pTS241(11)/pWG, and pTS265(1)/pWG into lacI(-) strain
  • Cloning pELa, pEL241, pEL265
    • Inoculation of dam- transformants w. pTS, pTS241(11), and pTS265(1)
  • Microscopy
    • No difference between pWG/pTS + IPTG and pWG/pTS - IPTG in TOP10 background

June 30 (Thur)

BioSketch CollinsMod group

  • Cloning pELa, pEL241, pEL265
    • Miniprepped dam- transformants
    • Digested w. XbaI/SphI to remove P(trc)-cI
    • Klenow rxns
    • Gel run shows that XbaI still doesn't cut
      • Single digests with XbaI and SphI shows that XbaI digest is uncut
  • Cloning pELc
    • Repeated blunt ligation of pTS[BS]1.7kb to pTS[BS]4kb
    • Transformation of ligation into TOP10
  • Clonig pWC
    • Received mCherry from MIT
    • Transformed into TOP10
  • Reconstituting Collins
    • Co-transformations of pWG/pTS, pWG/pTS241(11), pWG/pTS265(1) into lacI(-) successful

July

July 4 (Mon)

BioSketch CollinsMod group

  • Cloning pELc
    • No transformants from 2005-06-30 ligations
    • Repeated digests w. more DNA
  • Reconstituting Collins
    • Cultured pWG, pWG/pTS, pWG/pTS241(11), pWG/pTS265(1) in lacI(-)
  • Cloning pWC
    • Cultured mCherry in TOP10 for minipreps

July 5 (Tue)

Group Meetings

  • BioSketch group
  • BioWire group

BioSketch CollinsMod team

  • Cloning pELc, pEL241 and pEL265
    • Klenow rxns on pTS[BS]vec, pTS[BS]in, pTS241(11)[BS], pTS265(1)[BS]
    • PCR purify pTS[BS]vec
    • CIP pTS[BS]vec @ 50°C for 1h
    • Gel extract pTS[BS]4kb, pTS[BS]1.7kb, pTS241(11)[BS]1.7kb, pTS265(1)[BS]1.7kb
    • Freeze extracts
  • Cloning pWC
    • Miniprepped pCh
    • Analytical digest w. PstI
    • Primer arrived for PCR cloning
  • Reconstituting Collins
    • Miniprep pWG/pTS, pWG/pTS241(11), pWG/pTS265(1)
    • Analytical digests w. SpeI on those and pWG-only: positive results
    • Diluted and incubated in 5ml selective medium w. or w.o. 2mM IPTG
  • Cloning pEG, pEC
    • Ordered PCR cloning primers

July 7 (Thur)

BioSketch CollinsMod team

  • Cloning pELc, pEL241, pEL265
    • 1-2 transformants for each ligation
    • Inoculation for minipreps
  • Cloning pWC
    • PCR of pCh w. P16/17 worked
    • O.n. digest of pWG and pCh_PCR w. XmnI/EcoRI
  • Reconsituting Collins
    • pWG+pTS seems to be inducible by 24J/m2 UV
    • New UV induction experiments
  • Testing pWCI
    • Inoculated pWG + pWCIb and pWG-only

July 8 (Fri)

BioSketch CollinsMod team

  • UV-induction assay
    • Conditions:
Starter strains: pWG in MC4100 and pWG+pTS in MC4100

Day 1
1.1 Overnight incubation 1 colony + 5ml selective LB.

Day 2
2.1 Overnight incubation 100ul cells + 40ul 250mM IPTG + 5ml selective LB

Day 3
3.1 Pipet 25ul cells onto agar. Incubate @ 30C for 2h.
3.2 Irradiate both pWG and pWG+pTS with UV @ 0, 12, 24 J/m^2.
3.3 Pipet and scrape off cells into 5ml selective LB, without IPTG.
3.4 Incubate overnight @ 37C.

Day 4
4.1 Backdilute 100ul cells + 5ml selective LB.
4.2 Incubate in 37C shaker for 2h, until OD600 ~ 0.3
4.3 Spin down 1ml cells and resuspend in 25ul (pWG) or 50ul (pWG+pTS) LB
4.4 Spot slide w. 1ul suspension.

Objective: 100x, oil
Filter:
- Bright-field: DIA-ILL
- GFP: B-2E/C FITC

July 10 (Sun)

BioSketch CollinsMod team

  • Cloning pTV, pELc and derivatives
    • Enzymatic preparation of pTV and pELc vectors
    • Will use previously digested & purified pELc inserts
  • Cloning pWCh
    • Transformants from 2005-07-08 did not have correct bands
    • Enzymatic preparation for pWCh vector (pWG[XE]) and insert (pChPCR[XE])
  • Cloning pEG
    • Enzymatic preparation for pEG vector (pWG[EXa]) and insert (P(trc)PCR[EXa])

July 11 (Mon)

BioSketch CollinsMod team

  • Gel extraction - elute with 30 uL
    • pWG [XE] 4.5 kb
    • pTS [BS]a 4 kb (CIP, Klenow, gel)
    • pTS [BS]b 4 kb (gel only)
  • Ligations
    • pWCh
      • 2 ul pWG [XE] 4.5 kb + 6 ul pCh_PCR [XE]
      • - ve: 2 ul pWG [XE] + 6 ul ddH2)
      • Incubated 1 hr RT
    • pTV
      • 1 ul pTS[BS}b 4 kb
      • Incubated 5 min RT
    • pELc/pEL241/pEL265
      • 4 ul pTS[BS]a 4 kb + 4 ul pTS[BS]/pTS241/pTS265 1.7 kb
      • -ve 4 ul pTS[BS]a + 4 ul ddH2O
      • Incubate 1 hr RT
  • Transformations
    • Top10 Competent Cells
      • 6 ul pELc/pEL241/pEL265/-ve (AMP)
      • 4 uL pTV (Amp)
      • 0.5 ul pWCIb2 (Amp)
    • MC4100
      • 8 ul pWCh (Tet)
      • 8 ul -ve (Tet)
    • All transformations incubated at 37 C for 1 hr

July 12 (Tues)

BioSketch CollinsMod team

  • Results of yesterday's transformations
    • pTS[BS]b (pTV) had 1 colony
    • pWCIb2 had many colonies
    • pTS [BS]a -ve potentially has 1 colony - tiny
    • pWCh had 2 colonies
    • No colonies on pELc/pEL241/pEL265/-ve for pWCh
  • Ordered E. coli K12 ER2925 dam- cells from NEB (cam resistance)

July 13 (Wed)

BioSketch CollinsMod team

  • Low copy plasmid minipreps of pTS, pWG, pWCIb2, pWCh
  • pWCh9 was extremely purple when pelleted, pWCh10 was less so
  • Analytical digests of pWCh9, pWCh10 indicated correct bands
  • Analytical digests of pTV, pTS, pWCIb2 showed very little DNA - redid digestions overnight

July 14 (Thurs)

BioSketch CollinsMod team

  • Minipreps of ligations for pWCh that Yin did
    • Potentially many of them could be good ones - all had shades of purple, though not as intense as pWCh9
  • Will send pWCh off for sequencing today

July 15 (Fri)

BioSketch CollinsMod team

  • Collins strain arrived today - will test his construct with that
  • pWCh was streaked on plates, but no visible signs of purple can be seen

July 27 (Wed)

  • Experiment #7
    • 2-o.n. incubation reveals no difference between switches that have been UV-treated and those that have not been.
    • What to do?
  • Getting a FACS machine
    • Arranged a meeting on Thur to look at the flow cytometer @ the Bauer Center.
  • Cloning pTSGC
    • GFPmut3b-P(trc)_P(L*)-mCherry in pWG vector
    • PCR of P(L*)-mCherry from pWCh to include XmaI site @ P(L*) and BamHI site @ mCherry.
      • Primers: P36/37
      • Thermocycling:
        1. 94°C for 10'
        2. 94°C for 30
        3. 50°C for 30
        4. 72°C for 1'30
        5. Go back to step 2 for 29 additional times.
        6. 4°C for o.n.
      • PCR product: PmChPCR

July 28 (Thur)

  • Cloning pTSGC
    • PCR for P(L*)-mCherry is successful.
  • Sequencing
    • pWG
      • P31/35: GFPmut3b
      • P33/34: P(L*)
    • pEG(1-6)
      • P31/35: GFPmut3b
  • FACS machine

August

Aug 03 (Wed)

  • Testing Collins switch
    • Replicating Kobayashi et al.: Success!
    • Shutting off thermo-sensitive switches with heat: Success!
    • Shutting off thermo-sensitive switches with IPTG: Partial success.
    • Maintaining OFF state for thermo-sensitive switches: Failure.

September-November

  • 2005-Sep-08: lab notebooks are scanned; click on the name of a team member to see their notebook.
  • 2005-Oct-23 Sunday 4pm Biolabs 1058: jamboree preparation (Discussion).
  • 2005-Nov-05 & 2005-Nov-06: iGEM Jamboree
  • 2005-Sep-26: have a look at the Cambridge iGEM team's promotional brochure PDF