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BioSketch The bacterial sketch pad.

BioSketch The bacterial sketch pad. Chris Doucette, Thomas Noriega, Hing Eng Yves Wang, Jennifer Gao, Yin Li Group Meeting 2005-08-15. BioBricks Team. LacI Switch. Constructed J06961 – LacI (mut 241) switch with EYFP Failed to make J06962 – LacI (mut 265) switch with EYFP.

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BioSketch The bacterial sketch pad.

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  1. BioSketchThe bacterial sketch pad. Chris Doucette, Thomas Noriega, Hing Eng Yves Wang, Jennifer Gao, Yin Li Group Meeting 2005-08-15

  2. BioBricks Team

  3. LacI Switch • Constructed J06961 – LacI (mut 241) switch with EYFP • Failed to make J06962 – LacI (mut 265) switch with EYFP

  4. LacI (mut241) Switch • After overnight Growth:

  5. LacI (mut241) Switch • After 4 h in 40°C:

  6. LacI (mut241) Switch • Suspect that the ligation actually failed = • No cI production, no LacI gene • Supported by an analytical digest • Redid digestion and ligation • Analytical digest shows appropriate bands • Microscope lamp broken • Sending out for sequencing

  7. A Cautionary Tale • What I was trying to do: Use miniprep isolated from a ligation mixture to transform JM2.300 cells for experiments. • Mini Prep: Decided to use J06961 mini 4 J06962 mini 2

  8. When I transformed the JM2.300 cells with the minis they did not act as expected under the microscope. Confused, hurt, and betrayed I picked ten colonies from the transformation and looked at their plasmids: Minis 1, 2, 3, 5, 8, and 9 were CONTAMINATED Minis 4, 6, 7, and 10 Were GOOD

  9. Sadly, the other plasmid transformation did not yield any good results…

  10. MORAL • Beware of contaminated minis, especially if you plan on using them to transform bacteria that you are going to experiment on directly.

  11. CollinsMod Team

  12. What has been done: CollinsMod • Experimenting with UV irradiation of cells on plastic substrate • Replicating last week’s results with Collins Switch • Finding temperature at which ThermoSwitches are bistable • Examining apparent mutations in CI-repressed P(L*)

  13. Irradiating Cells on Plastic Substrate • Logic: • Eliminate the agar bits that might be mistaken for cells • Substrate: • 3-cm polysterene petri plates • Cells: • JM 2.300 cells with amp resistance • Experiment (UV killing curve): • Dilute OD600 = 0.5 cells 1:107. • Pipet 50 or 100ul cells into 3-cm dish; spread evenly. • Irradiate. • Pipet another 100ul LB into dish. • Resuspend cells and pipet onto standard selective LB plates; spread. • Grow overnight.

  14. UV (Killing) Curve

  15. JM 2.300 GFP reporter Collins – 6 J/m2 Collins – 0 J/m2 Collins – 12 J/m2 Collins – 48 J/m2 Collins – 24 J/m2 UV Induces Signal in Collins Switch Day 1: 37C Day 2: 2mM IPTG  Day 3: UV irradiation, o.n. @ 37C  Day 4: Assay

  16. Finding Thermo-Bistability • Experimental design: • Expected results: • If cells are incubated (Day 3) at a temperature that permits bistability (Tb), then • Cells previously incubated @ 30C would remain ON. • Cells previously incubated @ 40C would remain OFF. • In other words: the state of the cell at Tb should be history-dependent. Day 1 30C Day 2 30C (ON) 40C (OFF) Day 3 30, 32, 35, 37, 40C (stay ON?) 30, 32, 35, 37, 40C (stay OFF?)

  17. JM 2.300 GFP reporter 40C switch 37C switch 30C  Tb 30C  Tb 40C  Tb 30C  Tb 40C  Tb 30C  30C 40C  Tb ThermoSwitches Exhibit Bistability at 30°C 30C 32C 35-38C 37C 40C

  18. M1 M1 M1 M1 A Closer Look at Behavior at 30°C A Closer Look at Behavior at 30°C 40C Switch 30C Switch 23.7% 76.3% 23.0% 77.0% 30C  30C 37.5% 62.5% 42.1% 57.9% 40C  30C

  19. Mutations in CI-repressible PL* Promoter • pWG (GFP reporter) • "Correct" sequence w.r.t. file sent from Collins lab • Canonical polymerase-binding regions (-10, -35) • Noncanonical CI-binding site OL1 • pTS (Collins switch) • Mutations are in -10 TATA box and OL1 binding site. • Should make LacI expression weaker and CI repression stronger. P(L*) from pWG 5’-GCGTCCTGCTGATGTGCTCATTATAACCGCCAGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGT-3’ 3’-CGCAGGACGACTACACGAGTAATATTGGCGGTCACCATAAATACAGTTGTGGCGGTCTCTATTAAATAGTGGCGTCTACCAATAGACA-5’ P(L*) from pTS/241/265 TATCACCGCCAGTGGTA:OL1 OL3: TATCACCGCAGATGGTT 5’-GCGTCCTGCTGATGTGCTCAGTATCACCGCCAGTGGTATTTATGTCAACACCGCCAGAGATAATTTATCACCGCAGATGGTTATCTGT-3’ 3’-CGCAGGACGACTACACGAGTCATAGTGGCGGTCACCATAAATACAGTTGTGGCGGTCTCTATTAAATAGTGGCGTCTACCAATAGACA-5’ GTTGTGGCGGTCTCTAT :OL2 lacI -10: TAATAT -35: ACAGTT

  20. ThermoSwitches: Results and Implications • Turning ON: Weak • Basal level of reporter expression at 30C is weakly elevated (compared to OFF state). • Would a lower temperature further increase GFP inductin? • Does UV irradiation increase GFP induction above baseline? • Turning OFF: Strong • Reporter expression is well-suppressed by temp > 37C. • Maintaining state: Weak • Reporter expression is moderately history-dependent at 30C, with tendency to be ON. • Reporter expression is history-independent at 32C, with weak suppression of signal. • Are the promoters leaky? • Is CI activity reduced at 30C? • In a previous experiment, IPTG-based repression at 30C did not work well. Was this due to decreased LacIts sensitivity to IPTG or decreased CI activity at 30C?

  21. Achieving Better ON State o.n. @ 40C (OFF) Day 1: 0, 12, 24, 48 J/m2 Day 2: 4h @ 25C (ON?) 4h @ 30C (ON?)

  22. Achieving More Robust Bistability • Continue playing with environmental parameters? • Try 31C? …. • If a more complete ON state is achieved, perhaps we will see a difference between maintaining the OFF state and maintaining the ON state. • Alter the parameters of the underlying circuit? • Put the switch circuit on a medium copy plasmid? • Make sure that both the LacIts promoter and the CI promoter have canonical polymerase binding regions or stronger RBSs. • Is CI activity weak at 30C? • Is state maintained in the Collins switch at 30C?

  23. And now, the long-awaited …

  24. T-Shirt Design Orr & Yin

  25. BBa_RTeKN0 BBa_B2WTF! Promoter Pizza-Inducible RBS Reference NA; could possibly work … maybe BBa_C(rand # of choice) BBa_B6D1N0 CDS BioWiredSketch-a-Lator Terminator Strength 5.5; units NA Front + = …

  26. Harvard iGEM 2005 KX Oh Danny XOrr Scarecrow Number 2 Crouching Connie T. Rialto Chickwich Lady Glowing Green Yin MacDaddy Chris the Destroyer Hidden Hing Tech Support Pam, George Kit, Radhika Alain Ira, Sasha Back

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