BioWire Progress Report Week Nine

1
BioWire Progress ReportWeek Nine

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

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
• Retransforming into MC4100 (LacI-) cells, since
  they seem hardier than DH5alpha cells

Receiver OutputPropagation Component (J06008)


6
Sequencing

• Sent in completed Lux parts for sequencing
• Total of 9 parts sent in (29 sequences)
• 4 readable sequences
• 3 okay, 1 missing an RBS
• Parts are being rebuilt with new reporters
  anyways
• All others had issues with multiple priming

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

• Does the Receiver Test Construct fluoresce with
  addition of AHL?
• Input acyl-homoserine lactone (AHL)
• Output EYFP fluorescence
• I13272 (receiver)
• Produces luxR constitutively
• Has EYFP after promoter lux pR, which is
  upregulated by the luxR-AHL complex

9
Experiments AHL to receiver

• Experimental Design
• Grow up overnight cultures
• Backdilute to 0.1 OD600
• Add appropriate concentration of AHL (0, 15, 50,
  150, 500 nM)
• Incubate 1 hr
• Place on M9 agar slides and image
• Controls
• Positive Constitutive YFP producer
  (I14033E0430)
• Negative Untransformed MC4100 cells

10
Experiments AHL to receiver

• Results
• All controls worked as expected
• Fluorescence was observed at all levels of AHL
  (except 0 nM) under the GFP and YFP filters
• Fluorescence levels at different concentrations
  of AHL were qualitatively comparable

11
PHASE
GFP FILTER
0 nM AHL
15 nM AHL
500 nM AHL
12
Experiments Receiver Construct

• Conclusions
• All parts in the receiver construct are
  functional
• Fluorescence can be observed at even low levels
  of AHL

13
Experiments aTc to sender/receiver

• Does the AHL sender, when induced by aTc, cause
  the receiver to fluoresce?
• Input aTc to sender cells
• Output EYFP fluorescence of receiver cells
• J06001 (AHL sender part)
• Produces luxI, which in turn produces AHL, in
  response to aTC

14
Experiments aTc to sender/receiver

• Experimental design
• Grow up both types of cells overnight backdilute
  both to OD600 0.01
• Add 1.56 ug/mL aTc to sender cells and incubate
  all cells in 37C shaker for 3 hours
• Add different concentrations (same volume) of
  sender cells to tubes of 2mL of receiver cells
• 1,5,10,20x senderreceiver ratios
• Incubate mixtures in 37C shaker for 40min
• Controls
• 500nM AHLreceiver 20x mixture500nM AHL
  receiver
• - LBreceiver, senderreceiver w/o aTc,
  receiveraTc

15
Experiments aTc to sender/receiver

• Results
• Controls behaved as expected
• Fluorescence of sender/receiver aTc mixtures
  was not high relative to negative controls
  (aTc/tetR fluorescence)
• Potential explanations
• Sender is not producing enough AHL (need to have
  higher senderreceiver ratios)
• Not adding enough aTc/tetR promoter for inducible
  aTc expression
• Sender part defective (promoter bad or luxI
  coding part bad)

16
100X Phase
100X YFP
100X GFP
100X CFP
20x Sender to Receiver ratio
Positive Control (receiver AHL)
Positive Control (receiver sender AHL aTc)
17
Experiments aTc to sender/receiver

• Potential explanations
• Sender is not producing enough AHL (need to have
  higher senderreceiver ratios)
• Not adding enough aTc/tetR promoter for inducible
  aTc expression
• Sender part defective (promoter bad or luxI
  coding part bad)
• Promoter appears to be working (aTc inducible)

18
100X Phase
100X YFP
R0040/E0434 -aTc
R0040/E0434 aTc 1.56 ug/mL
19
Planned Experiments

• Replicating Sender/Receiver experiment
• Higher sender cell concentration
• Creating a time course for AHL induction with
  receiver construct
• Testing Propagation Constructs with new YFP (to
  be cotransformed with LuxR producers)

20
Photolithography

• Made 4 rounds of masters
• 90 micron really good uniformity (/- 10 um)
• Unknown, practice at 1mm protocol
• 4 wafers, 600 900 microns
• 1 mm
• Really good uniformity
• All features stayed on!
• PDMS and agarose
• Stamped from 100 micron and most recent 1mm.

21
150 micron master
8/2 150 micron, second round 85-110 micron
range
22
1mm master
8/5 1 millimeter, second round, 90
sec. exposure 715-975 micron range
870 um
910 um
905 um
890 um
970 um
875 um
945 um
955 um
725 um
790 um
725 um
795 um
715 um
780 um
715 um
775 um
715 um
23
Photolithography

• Issues in the cleanroom
• Still not getting perfectly level surfaces.
• Wafer still sticks to mask.
• Havent been able to spin a final coat for
  uniformity as the spinners have been down.
• Only other step requiring work is actual stamping
• Still not very precise can we blot?

24
Stamps
1mm wide lines
500 micron lines
1mm wide perimeter
25
Photolithography

• Practice stamping for precise cell growth
• A few more cleanroom cycles to increase stamp
  depth, fix final uniformity issues

26
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.

27
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) More experiments, construction with
  new reporters
• Week 10 (8/8)
• Week 11 (8/15)
• Week 12 (8/22)
• Week 13 (8/29)