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

• Rebuilt parts with new YFP reporters
• Experiments
• Constitutive senders AHL receivers
• AHL cotransformed receivers
• Sent parts in for sequencing
• Photolithography

3
Building the Circuits

• Rebuilt all major Lux parts with new YFP
  reporters
• Cotransformed AHL receiver with propagation
  constructs
• Nearly complete with major Las constructs

4
Building the Circuits

• Started building new circuit (thanks to Ira)
• Based off of repressilator transcriptional
  cascade of repressors
• Puts a time delay between AHL induction and CI
  repression, thus ensuring a pulse
• Parts were ordered from MIT within two cycles of
  completion

5
Building the Circuits

• Current Design
• CI, LuxI, and reporter induced at same time
• New Design
• Cascade causes CI to be induced later than LuxI
  and reporter, eliminating competition between
  propagation and repression

6
Experiments

• Constitutive Senders to Receivers
• Does the LuxI sender part work?
• How much LuxI is needed to activate the
  receivers?
• AHL to Cotransformed Receiver Constructs
• Can the contransformed constructs be induced by
  AHL?

7
Experiments Constitutive Senders

• Does the Receiver Test Construct fluoresce when
  combined with constitutive LuxI senders?
• Input Combining LuxI senders with receiver cells
  in varying ratios
• LuxI produces AHL, which binds with LuxR to
  activate LuxPR promoter
• Output Fluorescence

Constitutive promoter is Plambda, thanks Biosketch
8
Experiments Constitutive Senders

• Experimental Design
• Overnight cultures were backdiluted to 0.1 OD600
• Sender cells were backdiluted, IPTG induced for 2
  hours, then backdiluted again
• Sender and receiver cells were combined in
  varying ratios by volume
• 11 (senderreceiver), 21, 41
• Controls
• Positive AHL Receiver
• Negative Receivers alone
• Cells were imaged after 40 minutes of incubation

9
Experiments Constitutive Senders

• Results
• Positive control worked as expected
• Negative control showed a significant amount of
  background fluorescence observed under YFP and
  GFP filters
• Fluorescence was observed at all senderreceiver
  ratios under YFP and GFP filters

10
- control, 100X, GFP
- control, 100X, phase
11 ratio, 100X, GFP
11 ratio, 100X, phase
11
- control, 100X, GFP
- control, 100X, phase
21 ratio, 100X, GFP
21 ratio, 100X, phase
12
Experiments Constitutive Senders

• Conclusions
• Fluorescence seems qualitatively stronger with
  addition of senders, but still unsure if it is
  caused by senders or background
• LuxPR promoter (controls YFP) has weak
  constitutive expression noise problems with
  construct
• Complicated because senders and receivers are
  mixed unsure what percent of receivers are
  actually fluorescing
• Solid media may be better way of experimenting
• Using stamp, can separate senders and receivers
  while allowing for induction

13
Experiments Cotransformants

• Can LuxR producers cotransformed with receiver
  constructs respond to addition of AHL?
• Input AHL
• Output YFP fluorescence

On KAN plasmid
On AMP plasmid Degradation tags on YFP and LuxI
varied
Cotransformed in MC4100 cells (LacI-)
14
Experiments Cotransformants

• Experimental Design
• Positive Control Receiver Construct AHL
• Negative Control Cells without YFP AHL
• Negative Control No AHL added to cotransformants
• Experimental Strains
• J06007.4A LuxI (LVA), strong RBS, YFP (AAV-)
• J06007.4B LuxI (LVA), strong RBS, YFP (AAV)
• J06008.4A LuxI (LVA-), strong RBS, YFP (AAV-)
• J06008.4B LuxI (LVA-), strong RBS, YFP (AAV)

15
Experiments Cotransformants

• Experimental Design
• Overnight cultures were backdiluted to 0.1 OD600
• 500 nM AHL was added to each culture
• Cells were imaged after 40 minute incubation

16
Experiments Cotransformants

• Results
• Positive Control worked as expected
• Negative Control No YFP cells did not fluoresce
• Cells with YFP (AAV-) fluoresced even without AHL
  addition
• Cells with YFP (AAV) did not fluoresce even with
  AHL addition

17
Positive Control Receiver Test Construct
500 nM AHL
No AHL
18
J6007.4A LuxI (LVA), YFP (AAV-)
500 nM AHL
No AHL
19
J6007.4B LuxI (LVA), YFP (AAV)
500 nM AHL
No AHL
20
J6008.4A LuxI (LVA-), YFP (AAV-)
500 nM AHL
No AHL
21
J6008.4B LuxI (LVA-), YFP (AAV)
500 nM AHL
No AHL
22
Experiments Cotransformants

• Is system being triggered?
• Yes LVA tag on LuxI (produces more AHL to
  propagate signal) significantly reduces efficacy
  of AHL propagation LVA strain had significantly
  weaker fluorescence

23
Experiments Cotransformants

• AAV- YFP Positive feedback loop is causing
  auto-firing even in absence of AHL
• AAV YFP tag on YFP is too strong to visualize
  fluorescence (YFP gets degraded too quickly)?
• AAV YFP was visible in positive control
  (I13272), but that is under slightly different
  promoter (lux vs luxCI)
• Having propagation should only increase signal
• Need to build positive control with luxCI
  promoter

24
Experiments Cotransformants

• Weak constitutive activity of LuxPR is
  problematic
• Will test constructs with weaker RBS to reduce
  amount of noise
• Also test constructs with repressor

25
Planned Experiments

• Testing cotransformants with varying RBS
  strengths
• Testing receivers cotransformed with repressors
  (aka pulse generator)
• Testing senders with receivers on solid media
• Using the FACS for more accurate, quantitative
  measurements
• Using the wicked cool stamps

26
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.

27
150 micron master
8/2 150 micron, second round 85-110 micron
range
28
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
29
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?

30
Stamps
1mm wide lines
500 micron lines
1mm wide perimeter
31
Photolithography

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

32
This Week

• Building parts
• Continue cotransforming Lux test constructs
• Build revised circuit design
• Finish building Las parts with new reporters
• Experiments
• Test cotransformants with different RBS,
  repressor component
• FACS
• Solid media experiments
• Photolithography
• STAMP STAMP STAMP STAMP STAMP

33
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) More experiments, STAMP STAMP
  STAMP
• Week 11 (8/15)
• Week 12 (8/22)
• Week 13 (8/29)