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BioWire Progress Report Week Five. Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush. Continued building Lux and Las test constructs. Reorganized BioBrick information database. Got our mask back from Dr. Lu. Started pouring agarose stamps. - PowerPoint PPT Presentation
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BioWire Progress ReportWeek Five
Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush
Last Week
Continued building Lux and Las test constructs.
Reorganized BioBrick information database. Got our mask back from Dr. Lu. Started pouring agarose stamps. Ordered CFP filter.
Building the Circuits
Sasha created a REALLY HOT DATABASE OF LIGATIONS OMFG HOT!!
Higher success rate with transformations - now within 2 cycles of finishing all of our parts
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
Building
Completed
J06001: Ptet+luxI(LVA+)
J06002.x: Ptet+luxI(LVA-)
J06003: Ptet+ ECFP
J06000: LuxPL + luxR + luxPR + YFP
J06004.x (vary RBS)
Notes
F1611 missing.(R0040+B003x) + (C0161+B0015)
Building I13272 missing.(R0063+I0462) + (R0062+E0432)
Building
Done Lig./Trans. 7/9
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!
Building
Notes
Building
Building
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
(I14018 + (B0034+C0079+B0015 + R0079) ) = J06400
( I14018 + (B0034+C0079+B0015 + R0079) + E0432) = J06100
Building
Done Lig./Trans. 7/9
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
Mask– We have our mask. Cost: Free.– Next step: Ed (from Kit’s lab) will make us two
masters from the mask. Time frame: end of the week.
– Step after that: Go to Kit’s lab and make PDMS negative molds from the two masters.
Photolithography
Master– Because we will be stamping by hand, we really
want our features to have tangible depth. But SU8, the photoresist that we will use, is normally used up to 250 microns.
– We have a protocol from the Whitesides group for making features 1 mm deep, and we’re also in contact with cleanroom personnel about different types of SU8 and protocols.
Photolithography
Stamps– To make negative molds, we cut up a 96 well plate using a
heated spatula. This was done under a hood.– We placed the negatives into small beakers and poured 5%
agarose in PBS to make stamps. We separated the stamps from the molds.
– It turns out that the 96 well plates have tiny writing between wells to indicate the row and column. We found this accidentally by looking at the stamps, meaning that the agarose has clear definition at ~200 microns (or less?).
Photolithography
This Week
Parts scheduled to be finished today (still need analytical digestion for confirmation)
– J06004 - Receiver with Repressor Component– J06007 - LVA+ Receiver Construct with propagation– J06008 - LVA- Receiver Construct with propagation
Conduct experiments on Sender Reporter Test Construct with CFP filter
Plan and conduct experiments on Receiver Constructs Finish building lux, las constructs Practice inking with our stamps.
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): Test constructs, finish building parts, get master mold,
make PDMS molds Week 7 (7/18): Construct IFFL Week 8 (7/25): Test it Week 9 (8/1): “ Week 10 (8/8): “ Week 11 (8/15): “ Week 12 (8/22): “