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BioWire Progress Report Week Six “Halfway There”

BioWire Progress Report Week Six “Halfway There”. Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush. Last Week. [Almost] Finished building Lux parts Conducted experiments with Lux sender test construct Got cleanroom certification. Building the Circuits.

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BioWire Progress Report Week Six “Halfway There”

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  1. BioWire Progress ReportWeek Six“Halfway There” Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush

  2. Last Week • [Almost] Finished building Lux parts • Conducted experiments with Lux sender test construct • Got cleanroom certification

  3. Building the Circuits • Lux • All parts should be finished today (awaiting analytical digest). • Received DH5alpha cells for transformation • Started moving parts onto Kan plasmids in order to cotransform into cells. • Las • Still building parts. • Switched constitutive promoters, P(Bla)-->P(Cat) because the old part just wasn’t working. • Moved within two cycles of finishing.

  4. KAN: Receiver Repressor Component (J06004) BioWire Cell AMP: Receiver Output+ Propagation Component (J06007,8) Building the Circuits • Cotransformation Plan • Putting CI repressor on kan plasmid enables us to control the copy number, and thus repressor levels, through IPTG induction

  5. Sender Reporter (eCFP), Ptet Test J06003 Experiments • Testing the Sender Reporter Construct • Input: anhydrotetracycline (aTc) • Output: fluorescence (CFP)

  6. Experiments • Experimental Design • Positive Control - constitutive GFP (gift from Yin) • Negative Control - no aTc added/DMF only • Negative Control - strain without CFP (LuxI sender under Tet promoter)

  7. Experiments • Experimental Design • Grow up overnight cultures of cells • Backdilute to 0.1 OD600 • Add varying concentrations of aTc • 25 μg/ml, 12.5 μg/ml, 6.25 μg/ml, 3.13 μg/ml, 1.56 μg/ml, 781 ng/ml, 391 ng/ml, 195 ng/ml • Check for fluorescence after 3 hours using CFP filter on microscope

  8. Experiments • Results • Fluorescence was observed in the Sender Reporter Test Construct, visible with both CFP and GFP filters J06003: 3.13 ug/ml aTc, 100X, GFP filter J06003: 3.13 ug/ml aTc, 100X, CFP filter

  9. Experiments • Results • Cell density decreased with higher concentrations of aTc (same volume of aTc solution was added to all samples) • DMF only cells did not seem to be affected - is aTc toxic at higher concentrations?

  10. J06003: no aTc (DMF only), 100X, GFP filter without UV J06003: 1.56 ug/ml aTc, 100X, GFP filter J06003: 3.13 ug/ml aTc, 100X, GFP filter J06003: 6.25 ug/ml aTc, 100X, GFP filter

  11. Experiments • Results • No aTc added negative control did not fluoresce. • No CFP negative control fluoresced at levels comparable to experimental samples

  12. J06001: 1.56 ug/ml aTc, 100X, GFP filter J06003: 1.56 ug/ml aTc, 100X, GFP filter

  13. Sender Construct (LVA+): aTc -> AHL J06001 Experiments • Possible explanations for negative control fluorescence • Faulty part - we will be rebuilding and retesting; indication that we should be sequencing our finished parts • Autofluorescence

  14. Planned Experiments • Testing the AHL receiver • Testing the Weiss pulse generator contransformed with the receiver/receiver repressor construct • Testing the BioWire pulse propagator cotransformed with the receiver/receiver repressor construct

  15. Photolithography • Making the Master • Completed general cleanroom training. • Will get tool-specific training as soon as Kit’s lab gets their SU-8 2. • SU-8 2075 is being ordered this morning. (Balance of low viscosity and good thickness.) • Talked with Microchem researcher about SU-8 types and protocols.

  16. Photolithography • Stamps • Poured PDMS negative molds out of 33 rpm vinyl records in Kit’s lab. • Continued to experiment with pouring stamps from 96- and 384-well plates, using 3% and 5% high gel strength agarose. • Stamped cells using last week’s stamps.

  17. Stamp Picture

  18. This Week • Building parts • Test constructs for Lux, finish Las parts. • Cotransform finished Lux parts. • Part validation/sequencing. • Experiments • Test receiver constructs • Reconstruct and test LuxI sender (unexpected fluorescence) • Photolithography • Go into cleanroom to finish training and make a first master.

  19. Updated Schedule • Week 1 (6/6): Project Choice and Design • Week 2 (6/13): Got parts and set up tests • Week 3 (6/20): Began building test constructs, finished sender • Week 4 (6/27): Finish receiver, receiver w/repressor; CAD a mask • Week 5 (7/4): Continued building parts, received mask • Week 6 (7/11): Finished Lux, Tested senders, made PDMS molds • Week 7 (7/18): More experiments, finish Las; complete a cycle of making a master mold, PDMS mold, and stamp • Week 8 (7/25): Experiments • Week 9 (8/1): “ • Week 10 (8/8): “ • Week 11 (8/15): “ • Week 12 (8/22): “

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