Articles
https://doi.org/10.1038/s41589-020-0591-0
Acoustic biosensors for ultrasound imaging of
enzyme activity
Anupama Lakshmanan
1,5
, Zhiyang Jin
2,5
, Suchita P. Nety
1
, Daniel P. Sawyer
3
,
Audrey Lee-Gosselin
1
, Dina Malounda
1
, Mararet B. Swift
1
, David Maresca
1,4
and Mikhail G. Shapiro
1
✉
1
Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA, USA.
2
Division of Engineering and Applied Sciences,
California Institute of Technology, Pasadena, CA, USA.
3
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA,
USA.
4
Present address: Department of Imaging Physics, Delft University of Technology, Delft, Netherlands.
5
These authors contributed equally: Anupama
Lakshmanan, Zhiyang Jin.
✉
e-mail: mikhail@caltech.edu
SUPPLEMENTARY INFORMATION
In the format provided by the authors and unedited.
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ture CheMiCAL BioL
oG
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Lakshmanan
, Jin
et al.
Page
1
of
2
SUPPLEMENTARY INFORMATION
Ac
oustic Biosensors for Ultras
ound
Imaging of Enzyme Activity
Anupama Lakshmanan, Zhiyang Jin, Suchita P. Nety, Daniel P. Sawyer, Audrey Lee
-
Gosselin, Dina
Malounda,
Margaret B. Swift,
David Maresca
and Mikhail G. Shapiro.
Correspondence to:
mikhail@caltech.edu
CONTENTS
Suppleme
ntary Table 1
References for
Supplementary
Information
Plasmid
Name
Description and
Purpose
Transcript
ional
regulators
Output gene
product(s)
Insertions/Tags
(including
linkers)
Reference
Infor
-
mation
WT C
-
His Ana
GvpC in
pET28a
Ana
gvpC
used
as wild
-
type control for TEV
and calpain sensor
pT7, LacO
WT C
-
His Ana GvpC
SLE
-
His6 at
C
-
terminus
Addgene
ID# 85732
WT N
-
His Ana
GvpC in
pET28a
Ana
gvpC
used
as wild
-
type control for ClpXP
sensor
pT7, LacO
WT N
-
His
-
Ana
-
GvpC
G
-
His6
-
SG at
N
-
terminus
Addgene
ID#
153
294
C
-
His
-
GvpC
-
TEV
Ana
gvpC
with TEV
cleavage site
pT7, LacO
C
-
His Ana GvpC with
TEV cleavage site
SLE
-
His6 at C
-
terminus,
GSGSGSG
-
ENLYFQG
-
SGSGSG in GvpC
repeat 2
Addgene
ID# 153296
C
-
His
-
GvpC
-
Calpain
Ana
gvpC
with
calpain
cleavage site
pT7, LacO
C
-
His Ana GvpC with
calpain cleavage site
SLE
-
His6 at C
-
terminus,
GSGSG
-
QQEVYGMMPR
D
-
GSGSG in GvpC
repeat 2
Addgene
ID# 153295
N
-
His
-
GvpC
-
ssrA
Ana
gvpC
with ssrA
degradation tag
pT7, LacO
N
-
His Ana GvpC with
ssrA degradation tag
G
-
His6
-
SG at N
-
terminus,
SG
-
AANDENYALAA
at C
-
terminus
Addgene
ID# 153297
pBEST_OR2_
OR1
-
Pr
-
UTR1_ClpP
-
T500
clpP
plasmid for use in
the cell
-
free TX
-
TL
system
OR2
-
OR1
-
Pr
ClpP
Addgene
ID# 153302
pACYC
-
FLAG
-
dN6
-
His
clpX
p
lasmid for
use in
the cell
-
free TX
-
TL
system
pT7, LacO
Flag
-
ClpXdeltaNLinkedHe
xamer
-
His6
Flag tag
at
N
-
terminus
His6 at C
-
terminus
L20 linkers
Addgene
ID# 22143
pET28a_T5
-
ARG1
Original acoustic
reporter gene
construct (
ARG
WT
)
pT5, LacO
Ana GvpA, WT Ana
GvpC, Mega GvpR
-
U
Addgene
ID
# 106476
Lakshmanan
, Jin
et al.
Page
2
of
2
Supplementary
Table 1:
List and features of genetic constructs used in this study.
pKD3
1
was a gift from Barry L. Wanner (Addgene plasmid # 45604 ; http://n2t.net/addgene:45604 ;
RRID:A
ddgene_45604)
.
pKD46
1
was obtained from the Coli Genetic Stock Center
(CGSC,
https://cgsc.biology.yale.edu/Site.php?ID=64672
).
pE
-
FLP
2
was a gift from Drew Endy & Keith Shearwin
(Addgene plasmid # 45978;http://n2t.net/addgene:45978 ; RRID:Addgene_45978)
.
The
pBEST_OR2
-
OR1
-
Pr
-
UTR1_Clp
P
-
T500 was a gift from
Zachary Sun and
Richard Murray
3
.
pACYC
-
FLAG
-
dN6
-
His
4
was a gift from Robert Sauer (Addgene plasmid # 22143 ;
http://n2t.net/addgene:22143 ; RRID:Addgen
e_22143)
The
pTARA
backbone was modified to make the araBAD
-
BCD20
-
ClpP
-
BCD17
-
ClpX and pTEtR
-
BCD2
-
Ana GvpC constructs. pTARA
5
was a gift from Kathleen Matthews (Addgene plasmid # 31491;
http://n2t.net/addgene:31491 ; RRID:Addgene_31491)
.
REFERENCES
FOR
SUPPLEMENTARY INFORMATION
:
1.
Datsenko, K.A. & Wanner, B.L. One
-
step inactivation of chromosomal genes in Es
cherichia coli
K
-
12 using PCR products.
Proceedings of the National Academy of Sciences
97
, 6640
-
6645 (2000).
2.
St
-
Pierre, F. et al. One
-
step cloning and chromosomal integration of DNA.
ACS Synthetic Biology
2
,
537
-
5
41 (2013).
3.
Zachary, Z.S., Jongmin, K., Vipul, S. & Richard, M.M. Protein degradation in a TX
-
TL cell
-
free
expression system using ClpXP protease.
bio
Rxiv
(2015).
4.
Martin, A., Baker, T.A. & Sauer, R.T. Rebuilt AAA + motors reveal operating principles for ATP
-
fuelled machines.
Nature
437
, 1115
-
11
20 (2005).
5.
Wycuff, D.R. & Matthews, K.S. Generation of an AraC
-
araBAD promoter
-
regulated T7
expression sy
stem.
Analytical Biochemistry
277
, 67
-
73 (2000).
pET28a
-
T5
-
ASG_ClpXP
Acoustic sensor gene
for ClpXP (
ASG
ClpXP
)
pT5, LacO
Ana GvpA, dGvpC,
Mega GvpR
-
U
SG
-
AANDENYALAA
at C
-
terminus
Addgene
ID# 153299
pKD3
Frt
-
flanked cat cassette
for recombineering
CAT
Addgene
ID
# 45604
pKD46
Plasmid that
carries
the Lambda Red
recombineering system
pBAD,
araBAD
operon
Gam, Beta, Exo
Coli Genetic
Stock Center
pE
-
FLP
FLP
recombinase to
remove the integration
module in
reombineering
pE
FLP
Addgene
ID
# 45978
araBAD
-
BCD20
-
ClpP
-
BCD17
-
ClpX
Expression of
clpX
and
clpP
under araBAD
promoter
pBAD,
araBAD
operon
ClpX, ClpP
Addgene
ID # 153301
pTetR
-
BCD2
-
Ana GvpC
Wild
-
type
Ana
gvpC
under
Tet promoter
pTet, TetO
WT Ana GvpC
Addgene
ID # 153298