William E' Bentley

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Bacterial Quorum Sensing A Target for Systems
Bioengineering Research
William E. Bentley Herbert Rabin Distinguished
Professor Chair, Fischell Department of
Bioengineering
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What is quorum sensing bacterial crosstalk?
www.cobiotex.fr
Chong Yung, UMCP
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What does bacterial cell signaling do?
Fish, squid, light organ
Myxococcus
Paenibacillus vortex
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Immerse our device in the future
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Systems Bioengineering
I. Towards Understanding the Biology .
II. Towards Applications in Bioprocessing .
III. Towards Bio/Devices for Drug Discovery .
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I. Towards Understanding the Biology .
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Mechanism of quorum machinery in E. coli
LsrR
LsrC
ATP
LsrA
LsrK
LsrB
ADP
P
LsrD
LsrA
P
lsrG
lsrF
lsrB
lsrC
lsrD
lsrA
lsrR
lsrK
-

CRP
-
?s
-
cAMP
Glucose or PTS sugars
-
luxS
pfs
LuxS
E. coli
pfs
AI-2
SRH
SAH
Wang et al., J. Bact., 1872066-76, 2005.
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II. Towards Applications in Bioprocessing .
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Regulon Engineering / Global Targets and Tools
signal transduction
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Protein production curtails crosstalk .
that
can be restored
w/ conditioned media
1400
1200
AI-2 Activity
1000
800
600
400
200
OPH Induction
0
0
1
2
3
4
5
6
10
CAT Induction
CAT
OPH
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6
4
Specific OPH Activity (U/µg protein)
2
0
0
1
2
3
4
5
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Specific CAT Activity (U/µg protein)
Time (h.p.i.)
Time (h.p.i.)
Tsao et al., 2006 (submitted)
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III. Towards Bio/Devices for Drug Discovery .
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Microfabrication and Biotechnology
Biofabrication
Yi et al., Biomacromolecules, 6, 2881-94, 2005
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Biofabrication takes advantage of Natures
self-assembly and recognition capabilities
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Engineering the Interface Building on
Microfabrication w/ Polysaccharide Scaffold
chitosan
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Tyrosinase to Construct Protein-Chitosan
Conjugates
Chitosan
Enzymatic Assembly -
Tyrosyl residue
O
O
H
T
y
r
o
s
i
n
a
s
e
O
2
O
Protein-chitosan Conjugate
Protein
Activated Protein
Biopolymers 64 292 (2002) Biomaterials 24 2831
(2003) Biotech. Bioeng. 931207. (2006)
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Engineering the Interface Building on
Microfabrication
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In situ activation for patterned assembly
Electro-assembly of biopolymeric scaffold
Tyrosinase Activation for Protein Assembly
tyrosinase
GFP
GFP activation assembly
A
B
C
D
power supply
Chitosan deposition
All chips have deposited chitosan A GFP
Tyrosinase 4C 16h 5 sec exposure B GFP
Tyrosinase 30C 2h 30 sec exposure C GFP 4C 16h
30 sec exposure D Tyrosinase 4C 16h 30 sec
exposure
Lewandowski et al., 2006 (submitted)
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Assembling Proteins from Solution within Device
3-D
Park et al., Lab on a Chip, 2006
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Assembling Proteins from Solution within Device

Park et al., Lab on a Chip, 2006
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Biochemical Pathway for AI-2 Synthesis and Uptake
Protein Engineering add activatable pro-tag
Enzymatic Assembly of Chitosan
Conjugate and Directed (Signal Guided) Assembly
onto Surface
Lewandowski et al., 2007 (submitted)
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Magnetic Nanofactories AI-2 Synthesis and
Delivery
Fernandes et al., Metabolic Eng., 2006
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Building Magnetic Nanofactories
Fernandes et al., Metabolic Eng., 2006
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Testing Magnetic Nanofactories
c
d
Fernandes et al., Metabolic Eng., 2006
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III. Bio/Device Conclusions Applications small
molecule synthesis investigating genetic
circuits for systems biology microscale
biomanufacturing (programmable enzyme /
nucleic acid / protein assemblies) screening for
medical applications Pro-drug nanofactories
for delivery
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Many Thanks!
Matt DeLisa (Cornell), Shannon Kramer (Baylor),
Ranjan Srivastava (UConn), Liang Wang, Chen-Yu
Tsao, Yoshifumi Hashimoto (U. Florida), Li Yang,
David Small (EPA), Jun Li, Songhee Kim, Rohan
Fernandes (UMCP), Angela Lewandowski (UMCP), John
March (Cornell), Amanda Chambers, Laura Carpin,
Karen Carter, Hyunmin Yi Dr. Chi-Fang Wu (US
Army) and Emily Clough (CAB, Microarray) Dr. B.
Bassler (Princeton) and Dr. F. Baneyx
(Washington, Quorum Sensing) Dr. Govind Rao
(UMBC, GFP Optical Probes) Dr. Wolfgang Losert
(UMCP, Optical Tweezers) Dr. Tom Wood (UCONN
Quorum Sensing) Drs. Greg Payne, Gary Rubloff,
Reza Ghodssi (UMCP, UMBI, Chitosan BioMEMS) Dr.
James Valdes (US Army) Center for Biosystems
Research and UMBI U.S. Army, Chesapeake PERL, RWD
Deutsch Foundation, MIPS, Merck, DOE, and NSF
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Some philosophy for a University of Maryland
Engineer
BEHOLD THE TERRAPIN, HE MAKES PROGRESS ONLY
WHEN HE STICKS HIS NECK OUT
Dr. Robert E. Fischell
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Thanks for your time! Questions?