Engineered PostTranslational Logic PTL

1
Engineered Post-Translational Logic (PTL)
Samantha C. Sutton ?, Sara E. Neves ?, Lauren W.
Leung ?, and Drew Endy ?
? Division of Biological Engineering and ?
Department of Biology, Massachusetts Institute of
Technology
.
Abstract
Modeling as a Design Tool
Building and Testing a PTL Device
Example PTL Device Flip-Flop
Current synthetic biological circuits make use of
protein-DNA and RNA-RNA interactions to control
gene expression in bacteria. Systems that rely on
the regulation of gene expression are relatively
slow and unsuitable for many applications. Here,
we describe our work to engineer synthetic
biological systems in yeast using
post-translational modifications of proteins to
define system state and control cell function
such systems should have faster performance time
and enable a wider range of applications. We have
specifically chosen to focus on building
phosphorylation-driven protein circuits. We
modeled a specific instance of a
post-translational circuit using methods such as
Lyapunov exponents, and showed that the circuit
should behave as desired within a large parameter
space. We developed a set of peptide tags that
can be used to drive the phosphorylation of a
chosen substrate by a desired mitogen-activated
protein kinase (MAPK). Each phosphorylation event
alters a substrate output activity, such as
translocation, degradation, or other binding
event. These tags were developed using the
Phospholocator a construct whose
phosphorylation-mediated translocation is
controlled by MAPK activity. Specifically, MAPK
phosphorylation of the Phospholocator nuclear
localization sequence (NLS) controls recognition
of the NLS by cellular import machinery. The
Phospholocator serves three purposes to
determine the docking sites of MAPKs of interest,
to measure the in vivo activity of such MAP
Kinases, and to serve as a first set of
post-translational logic parts. Currently, we
have built a version of the Phospholocator that
is targeted by Cdc28 our next step is to build
Fus3-, p38-, and Hog1-activated instances.
Necessary Components of a PO4-MAPK Part
MAPK adds phosphate group here
MAPK binds here
In1
Out1
In2
Out2
State 2
State 1
In1
In2
Out1
Out2
When unphosphorylated Import machinery binds the
NLS and brings the device into the nucleus. When
phosphorylated Import machinery cannot bind the
NLS, and thus the device remains in the cytosol.
0
hold
hold
0
0
1
0
1
0
1
1
0
1
1
not allowed
The Phospholocator
Differences between PDL and PTL Flip-Flops
YFP ?-gal
PDL Flip-Flop
PTL Flip-Flop

• Uses of the Phospholocator
• To build sets of post-translational logic (PTL)
  parts.
• To determine the docking site and p-motifs of MAP
  Kinases of interest.
• To detect the activity of MAP Kinases.

Why Post-Translational Logic?
Our Goal Engineering Biology
Cell-Cycle Dependent Localization in Yeast

• Unlimited species concentrations
• Use hill coefficient to describe cooperativity
• Can make Pseudo-steady state assumption about
  protein-DNA binding

• Capped species concentrations
• Must generate cooperativity in new ways
• Cannot make pseudo-steady state assumption
  anywhere.

Physics
Electrical Engineering
Flip-Flop Model
Pheromone arrest (G1/S) nuclear
Nocodazole arrest (G2/M) cytosolic
Synthetic Biology

• Cdc28-Cln2 is active during late S and G2/M phase
  in yeast. In cells arrested with nocodazole,
  Cdc28 should be active, and phosphorylate the
  Phospholocator. The Phospholocator should then be
  cytosolic.
• Cdc28-Cln2 is inactive during G1 phase in yeast.
  In cells arrested with pheromone, Cdc28 should be
  inactive, and unable to phosphorylate the
  Phospholocator. The Phospholocator should then be
  nuclear.

Biology

• A and B are active until doubly phosphorylated
  by the other.
• Non-processive phosphorylation gives rise to the
  requisite ultrasensitive behavior of pink and
  green proteins
• Conservation of species means that we are dealing
  with a 4-D system.

Types of Intracellular Circuits

• Protein-DNA (Transcriptional) logic (PDL)
• Engineered around gene expression
• Easier to engineer
• Slow response time (hours)
• Uses one subset of cellular functions
• Post-translational logic (PTL)
• Engineered around protein modifications
• Difficult to engineer
• Fast response time (seconds)
• Explores new set of applications

PTL Flip-Flop is Robust to Parameter Fluctuations
Verification of Phosphorylation
Nocodazole



Pheromone

• We ran a SDS-PAGE gel of crude yeast lysate from
  cells arrested with nocodazole or pheromone.
• The Phospholocator was detected using anti-GFP
  antibody (gift from Bob Sauer).
• Phosphorylated construct runs slower than
  non-phosphorylated construct.

Phospholocator-PO4
An Example of a PTL Device
Phospholocator

• We used Matlab to vary k1, k2, k3, k4 over
  biologically relevant values, and then used
  fsolve to locate the fixed points. Shown above is
  the number of fixed points obtained for different
  values of k2 and k4 (k1 k3 10-4 (nM s)-1).
• Three fixed points can indicate a functional
  flip-flop, while one cannot.
• We computed the Jacobian of the system evaluated
  at each fixed point, and determined the
  corresponding eigenvalues.
• Two fixed points are asymptotically stable
  because they have all negative eigenvalues.
• The remaining fixed point is an unstable fixed
  point because it has one positive and three
  negative eigenvalues, indicating it has 3D stable
  and 1D unstable manifolds.

Conclusions

• We have shown that a PTL flip-flop will
  theoretically behave as expected over a wide
  range of parameter values.
• We have specified a system of PTL based on MAPKs
  and translocation
• We have designed a testing scaffold for
  identifying and characterizing docking and
  phosphorylation motifs, and are working on a
  first set of motifs.
• We have built a working instance of a PTL device
  the Phospholocator

Choice of Modification and Enzyme
PTL Flip-Flop is Robust to Concentration
Fluctuations

• Modification of choice phosphorylation
• Best studied phospho-mediated functions

Future Directions

• Build a Fus3 activated instance of the
  Phospholocator
• Build a simple inverter
• Develop a transcription-based localization assay
  for directed evolution of motifs

.
Acknowledgements

• Our three stable points define a 2D plane in 4D
  space. We transformed coordinates so the plane
  was perpendicular to two axes, and thus we could
  work in two dimensional space.
• Varying initial concentrations of the two
  kinases, we measured the ratio of the change in
  initial concentration to the change in
  equilibrium concentration. This is known as the
  method of Lyapunov exponents. Larger ratios
  indicate a separatrix, which is the boundary of a
  domain of attraction.
• We can use this map to determine
• The range of concentrations over which our
  flip-flop will hold state.
• The amount of stimulus needed to switch states,
  or flip.

_at_Cambridge Pam Silver, Mike Yaffe, Doug
Lauffenburger, Gerry Sussman, the Endy lab, the
Bob Sauer lab, the Chris Kaiser Lab, the Steve
Bell Lab . _at_Berkeley Alejandro Colman-Lerner,
Jeremy Thorner, Kirsten Benjamin, Richard Yu,
Roger Brent, Gustavo Pesce Funding Howard Hughes
Medical Institute, National Institute of Health,
Merck Co., Inc. Our Goal Images
fromhttp//chemcases.com/cisplat/ cisplat01.htm
http//www.nature.com/nsu/030421/
030421-14.html http//www.northern.wvnet.edu/tda
nford/ icons/CELL.JPG Ricarose
Roque Transcriptional Modeling example from
Gardner et al, Nature. 2000 Jan
20403(6767)339-42.

• Enzyme of choice MAP Kinase
• Signaling pathways
• Well-studied
• Yeast has two well-known MAPKs Fus3, and Hog1
• Examples of modular MAPK docking sites