of 10
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1
Directed Evolution of Acoustic Reporter Genes
Using High
-
Throughput Acoustic Screening
Authors:
Robert C. Hurt
1#
, Zhiyang Jin
2#
, Mohamed Soufi
3
, Katie K. Wong
3
, Daniel P. Sawyer
1
, Hao K.
Shen
3
, Przemysław Dutka,
1,3
Ramya Deshpande
3
, Ruby Zhang
3
, David
R. Mittelstein
2
, Mikhail
G. Shapiro
2,3,4,*
Affiliations:
1
Division of Biology and Biological Engineering
2
Division of Engineering and Applied Science
3
Division of Chemistry and Chemical Engineering
4
Howard Hughes Medical Institute
#
Equal contribution
Ca
lifornia Institute of Technology
Pasadena, CA, USA 91125
*Correspondence should be addressed to MGS:
Email: mikhail@caltech.edu
Phone: 626
-
395
-
8588
Address: 1200 E. California Blvd, MC 210
-
41, Pasadena, CA 91125
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2
Figure S1. Detailed diagram of the
A
coust
ic
P
late
R
eader workflow.
(A) GVs are expressed
in
E. coli
as colonies on Petri dishes for 48 hr at 37°C, then colonies are picked into LB and grown
to saturation in liquid culture for 24 hr at 37°C. These saturated liquid cultures are then diluted
1:100 i
nto autoinduction LB and expressed for 24 hr at 37°C in 500 uL cultures in deep
-
well 96
-
S
3
well plates (square wells
are
used for maximum culture aeration; USA Scientific 1896
-
2800).
Aliquots of these cultures are aliquoted into an un
-
skirted 96
-
well PCR plat
e for subsequent
loading into phantoms. (B) A solution of 2% Ultrapure Agarose (Invitrogen, 16500500) is prepared
in 1X PBS and incubated at 60
°
C for at least 12 hr to degas. Agarose phantoms are then made
by pouring 75 mL of this solution into a 96
-
well p
hantom mold and incubating at 4
°
C for 10 min.
(C) A solution of 1% low
-
melting
-
temperature agarose (Goldbio, A
-
204
-
100) is prepared in 1X
PBS and incubated at 60
°
C for at least 12 hr
to degas. This solution is then aliquoted into an un
-
skirted 96
-
well PCR plate to be used for phantom loading. (D) Phantoms from B are loaded by
placing the 96
-
well PCR plates from A and C into 96
-
well heat block
s
at 42
°
C, and combining
equal volumes of c
ulture and agarose before pipetting into the empty phantom. (E) Phantoms
from D are scanned using the acoustic plate reader, which generates US data for each sample
and can image up to 12 96
-
well phantoms in a single scan.
Figure S2. Details of
gvpA
/
gvpB
mutant library construction.
(A) Overview of workflow for
creating either scanning site saturation or recombination libraries. (B) Details of library assembly
via a Golden Gate
-
based version of cassette mutagenesis (see Methods).
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4
Figure S3. Charac
terization of the top mutants from Round 1 of evolution.
(A
-
B) xAM
difference SBR as a function of pressure for each of the top mutants. N=4 biological samples
(each an average of 3 technical replicates). (C
-
D) OD600 measurements for the mutants shown
in A
-
B. N=4 biological samples.
S
5
Figure S4. Characterization of the top mutants from Round 2 of evolution.
(A
-
B) xAM
difference SBR as a function of pressure for each of the top mutants. N=4 biological samples
(each an average of 3 technical replicates). (C
-
D) OD600 measurements for the mutants shown
in A
-
B. N=4 biological samples.
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6
Figure S5. Acoustic collapse pressure curves for the best mutants identified in this study.
Acoustic pressure was applied
using
a parabolic Bmode
pulse
sequence with increasing
voltages,
and nonlinear signals were acquired
using
xAM at
3.1
MPa after each voltage step.
(A
-
B) xAM
acoustic collapse pressure curves for the top
-
performing mutants identified in this study. (C
-
D)
Data from A
-
B normalized to the same min and max. N=4 bi
ological samples.