BioWire Progress Report Week Six “Halfway There”
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Transcript of BioWire Progress Report Week Six “Halfway There”
BioWire Progress ReportWeek 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
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.
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
KAN: Receiver Repressor Component (J06004)
AMP: Receiver Output+Propagation Component (J06007,8)
BioWire Cell
Experiments
Testing the Sender Reporter Construct
– Input: anhydrotetracycline (aTc) – Output: fluorescence (CFP)
Sender Reporter (eCFP), Ptet Test
J06003
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)
Experiments
Experimental Design– Grow up overnight cultures of cells– Backdilute to 0.1 OD600– Add varying concentrations of aTc
25 ug/ml, 12.5 ug/ml, 6.25 ug/ml, 3.13 ug/ml, 1.56 ug/ml, 781 ng/ml, 391 ng/ml, 195 ng/ml
– Check for fluorescence after 3 hours using CFP filter on microscope
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
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?
J06003: no aTc (DMF only), 100X, GFP filter without UV J06003: 195 ng/ml aTc, 100X, GFP filter
J06003: 3.13 ug/ml aTc, 100X, GFP filter J06003: 6.25 ug/ml aTc, 100X, GFP filter
Experiments
Results– No aTc added negative control did not fluoresce.– No CFP negative control fluoresced at levels
comparable to experimental samples
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
Sender Construct (LVA+): aTc -> AHL
J06001
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
Photolithography
Master– Completed general cleanroom training.– Will get tool-specific training as soon as Kit’s lab
gets their SU-8 2.– Ordered (will order?) SU-8 2075. (Balance of low
viscosity and good thickness.)– Talked with Microchem researcher about SU-8
types and protocols.
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.
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 make a master.
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, make first
master/PDMS/stamp Week 8 (7/25): Experiments Week 9 (8/1): “ Week 10 (8/8): “ Week 11 (8/15): “ Week 12 (8/22): “