BioWire Progress Report Week Six “Halfway There” Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang- Xing Jin, Danny Popper, Sasha Rush
BioWire Progress Report Week Six “Halfway There”
Jan 13, 2016
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 Did experiments with Lux senders Got cleanroom certification. Building the Circuits. Lux - PowerPoint PPT Presentation
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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 Did experiments with Lux senders Got cleanroom certification
Building the Circuits
Lux– All parts finished (except receiver test construct).– Started moving plasmids in order to cotransform.
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.
Experiments
Lux System ConstructsSender Construct (LVA+): aTc -> AHL
J06001
Sender Construct (LVA-): aTc -> AHL
J06002
Sender Reporter (eCFP), Ptet Test
J06003
Receiver Test Construct: AHL -> YFP
J06000
Receiver Repressor Component: AHL -> cI
J06004
Receiver Construction Intermediate:
J06006
Receiver Output Component (Weiss):AHL -> YFP, cI repressible
J06005
Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+
J06007
Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA-
J06008
Lux System Constructs: Status
Receiver Test Construct: AHL -> YFP
Sender Construct (LVA+): aTc -> AHL
Sender Construct (LVA-): aTc -> AHL
Sender Reporter (eCFP), Ptet Test
Receiver Repressor Component: AHL -> cI
Construct Status
Completed
Done
Completed
J06001: Ptet+luxI(LVA+)
J06002.x: Ptet+luxI(LVA-)
J06003: Ptet+ ECFP
J06000: LuxPL + luxR + luxPR + YFP
J06004.x (vary RBS)
Notes
(R0040+B003x) + (C0161+B0015)
Building I13272 missing.(R0063+I0462) + (R0062+E0432)
Done
Done Lig./Trans. 7/15, miniprep today
F2621 initially missing.([(R0063+I0462) + R0062] + [B003x + (C0051+B0015)])
Lux System Constructs: Status
Receiver Output Component (Weiss):AHL -> YFP, cI repressible
Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+
Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA-
Receiver Output Construction Intermediate:
Construct Status
NO!
Done
Notes
Done
Done
J06006
(R0065+B0034) + E0032
(R0065+B0034) + (E0032 + B0015)
J06005
J06007.x
J06008.x
(((R0065+B0034) + (E0032 + B003x)) + (C0161 +B0015))x=1,2,4
(((R0065+B0034) + (E0032 + B003x)) + (C0061 +B0015))x=1,2,4
Las System Constructs
Receiver Test Construct: AHL -> YFP
Sender Construct (LVA+): aTc -> AHL
Sender Construct (LVA-): aTc -> AHL
Receiver Repressor Component: AHL -> cI
Receiver Construction Intermediate:
Receiver Output Component (Weiss):AHL -> YFP, cI repressible
Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+
Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA-
Sender Reporter (eCFP), Ptet Test
Las System Constructs: Status
Receiver Test Construct: AHL -> YFP
Sender Construct (LVA+): aTc -> AHL
Sender Construct (LVA-): aTc -> AHL
Sender Reporter (eCFP), Ptet Test
Receiver Repressor Component: AHL -> cI
Construct Status
Building
Building
Completed
J06101: Ptet+lasI(LVA+)
J06102.x: Ptet+lasI(LVA-)
J06103=J06003: Ptet+ ECFP
J06100: PBla + lasR + PLasR + YFP
J06104.x (vary RBS)
Notes
(R0040+B0034) + (C0178+B0015)
Building
(I14033 + (B0034+C0079+B0015 + R0079)) = J06400(I14033 + (B0034+C0079+B0015 + R0079)) + E0432 = J06100
Building
Done Lig./Trans. 7/15, miniprep today
J06400 + ( B003x+C0051+ B0015)x=4,1,2
(R0040+B0034) + (C0078+B0015)
Las System Constructs: Status
Receiver Output Component (Weiss):AHL -> YFP, cI repressible
Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA+
Receiver Output+Propagation Component: AHL-> {YFP, AHL}, cI repressible, LVA-
Receiver Output Construction Intermediate:
Construct Status
NO!
Building
Notes
Building
Building
J06106
((I14016 + B0034) + E0032)
J06105
J06107.x
J06108.x
x=1,2,4(I14016+B0034+E0032+B003x) + (C0178 + B0015)
x=1,2,4(I14016+B0034+E0032+B003x) + (C0078 + B0015)
(I14016 + B0034) + (E0032+ B0015)
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.
Stamp Picture
This Week
Building parts– Test constructs for Lux, finish Las parts.
Experiments
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): “
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