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BioWire Progress Report Week Four. Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush. Preliminary tests of LuxI aTc-induced sender reporter Continued building Las, RhI circuits and Lux receiver test constructs Finished CAD design for bacterial stamp

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Biowire progress report week four

BioWire Progress ReportWeek Four

Orr Ashenberg, Patrick Bradley, Connie Cheng, Kang-Xing Jin, Danny Popper, Sasha Rush


Last week

Preliminary tests of LuxI aTc-induced sender reporter

Continued building Las, RhI circuits and Lux receiver test constructs

Finished CAD design for bacterial stamp

Continuous modeling

Last Week


Weiss circuit no propagation

tetR promoter

LuxI

LuxPL

LuxPR

LuxR

cI

LuxBox (Lux/CI) hybrid promoter

YFP

Weiss Circuit (no propagation)

Sender

AHL

Receiver


Propagating pulse generator

LuxPL

LuxPR

LuxR

cI

LuxBox (Lux/CI) hybrid promoter

YFP

LuxI

Propagating Pulse Generator

tetR promoter

Sender

LuxI

AHL

Receiver


Sender reporter test construct

tetR promoter

eCFP

Sender Reporter Test Construct: aTc in, eCFP out

Part BBa_J06003

Sender Reporter Test Construct

  • Inducible by aTc (anhydrotetracycline)

  • Expresses eCFP (part #: BBa_E0422)

  • Used GFP filter for preliminary test; CFP expected to arrive last Friday


Preliminary pictures of srtc
Preliminary pictures of SRTC

SRTC + aTc (0.2 µg / mL)

SRTC - aTc

Oil immersion microscopy, 100x objective, GFP filter


Building the circuits
Building the Circuits

  • Second round of ligations mostly failed

    • exposed too long to UV during gel extraction?

  • I0462 (LuxR BioBrick) did not digest properly

    • Analytical digest worked fine

    • Repeating digestion as well as picking new colony, in case miniprep of I0462 is bad

    • May also try to digest differently, in case enzyme is the culprit

  • Repeated on Thursday


Photolithography
Photolithography

  • Finished mask design.

  • More?


Continuous model
Continuous Model

  • Received MATLAB files from David Braun, which model intracellular pulse generation (but not propagation)

  • Deciphered rate constants (but not units)

  • During downtime, we are extending this model to use positive feedback and to be intercellular rather than intracellular


This week
This Week

  • Finish receiver test constructs and begin experiments

    • J06000 -- receiver should be finished today

    • J06004 -- receiver w/repressor should be ready in 3 days

  • Finish las test constructs

  • Conduct experiments on SRTC with CFP filter


Updated schedule
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): Test component circuits; build stamps, test

  • Week 6 (7/11): Test cell signaling density

  • 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): “


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