BioWire Progress Report Week Eight

1
BioWire Progress ReportWeek Eight

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

TODO 4 I14033/E0434 images 6 Sequencing numbers
(X parts) 10 Images for AHL-gtreceiver
experiment 12 finish up atc sender/receiver
experiment Add atc r40/e434 experiment
2
Last Week

• Determined that our YFP reporter (used in the
  receiver) is defective
• Re-ordered a working YFP and began reconstruction
  using the new YFP
• Received the receiver test construct from
  BioBricks, conducted experiments
• AHL to receiver
• aTc to senderreceiver
• Sequencing results
• Photolithography

3
Oh YFP

• YFP Biobrick Parts
• E0030 (YFP, no degradation tags)
• E0032 (YFP, LVA)
• E0034 (YFP, AAV)
• We had been using E0032, since we want
  degradation of YFP for temporal analysis
• Turns out that E0032 does not glow when added to
  a constitutive promoter (degradation tag too
  strong?)
• Reporter control experiments

4
100x Phase
100x YFP Filter
I14033 E0430 Const. P(cat) YFP
I14033 E0434 Const. P(cat) YFP AAV
I14033 E0432 Const. P(cat) YFP LVA
5
(Re)Building the Circuits

• Now rebuilding circuits with E0030 (YFP), E0034
  (YFP AAV), and mCherry (LVA and LVA-)
• Thanks Biosketch
• Lux and Las components with YFP, YFP AAV, and
  mCherry LVA- will be ligated together today
• Also building mCherry sender reporter

Receiver OutputPropagation Component (J06008)

6
Sequencing

• Sent in completed Lux parts for sequencing
• Total of X parts (29 primers) sent in
• 4 readable sequences
• 3 okay, 1 missing an RBS
• Parts are being rebuilt with new reporters
  anyways
• All others returned more than 1 sequence (a lot
  of background)

7
Experiments

• AHL to receiver
• Is AHL working?
• Is the AHL receiver promoter working?
• aTc to senderreceiver
• Is the sender (AHL producer) working?
• Is aTc induction working (the tetR promoter)?
• Is luxI producing AHL?
• Solid media

8
Experiments AHL to receiver

• AHL receiver part (I13272/J06000)
• Produces luxR constitutively
• Has EYFP after promoter lux pR, which is
  upregulated by the luxR-AHL complex

9
Experiments AHL to receiver

• Add AHL to receiver cells do they glow?
• Protocol
• Grow up overnight backdilute to OD600 0.1
• Add AHL solution to cells at appropriate
  concentrations
• Negative controls 0 AHL, AHL cells w/o
  receiver
• Positive controls none
• Incubate in 37C shaker for 40min
• Image

10
Experiments AHL to receiver

• Add AHL to receiver cells do they glow?
• Yes!

11
Experiments aTc to sender/receiver

• AHL sender part (J06001)
• Produces luxI, which in turn produces AHL, in
  response to aTC

12
Experiments aTc to sender/receiver

• Add aTc to sender cells, then mix with receiver
  cells do receiver cells glow?
• Protocol
• Grow up overnight backdilute sender to OD600
  0.01, receiver to OD600 0.1
• Add aTc to sender cells and incubate in 37C
  shaker for 3 hours
• Add different concentrations (same volume) of
  sender cells to fixed volumes of receiver cells
• Incubate mixture in 37C shaker for 40min

13
Photolithography

• Made a 5 µm master and two 150-350 µm masters
  (more on that later).
• Made PDMS negatives.
• Learned how to plasma oxiginate PDMS in order to
  reduce air bubble distortion.
• Made agarose stamps from the PDMS.

14
Photolithography

• Issues in the cleanroom
• Non-uniformity in photoresist
• caused wafer to bake onto the mask
• changed our soft-bake and development times
• and caused variations in feature height between
  150 µm and 350 µm. (We were aiming for 150 µm
  features.)
• Also, the agarose stamps we made had some minor
  issues (levelling, user error).

15
Photolithography

• With current PDMS negatives, will remake agarose
  stamps to start stamping cells.
• Will remake 150 µm master to increase uniformity.
• Will begin process of making 1mm master.

16
This Week

• Building parts
• Test constructs for Lux, finish Las parts.
• Move finished Lux parts onto DH5alpha cells.
• Part validation/sequencing.
• Experiments
• Test receiver constructs
• Reconstruct and test LuxI sender (unexpected
  fluorescence)
• Photolithography
• Go into cleanroom to make 150?m and 1000?m master.

17
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, eating pizza
  courtesy of Alain
• Week 8 (7/25) More experiments, Meeting Their
  Master
• Week 9 (8/1)
• Week 10 (8/8)
• Week 11 (8/15)
• Week 12 (8/22)
• Week 13 (8/29)