of 23
S1
Supporting Information
Quantitative Real-Time Analysis of Living Materials by Stimulated
Raman Scattering Microscopy
Chenxi Qian†
, Hanwei Liu†, Priya K. Chittur, Rahuljeet S. Chadha, Yuxing Yao, Julia A.
Kornfield, David A. Tirrell* and Lu Wei*
Division of Chemistry and Chemical Engineering, California Institute of Technology,
Pasadena, California 91125, United States
† These authors contributed equally: Chenxi Qian, Hanwei Liu
‡Current affiliations: Department of Medical Biophysics, University of Toronto, Toronto,
Ontario M5G 1L7 Canada; Physical Sciences, Sunnybrook Research Institute, Toronto,
Ontario M4N 3M5 Canada; Ontario Institute for Cancer Research, Toronto, ON, Canada
*Corresponding authors. Email:
tirrell@caltech.edu;
lwei@caltech.edu
Supporting Information
includes:
Figs. S1 to S12
Tables S1 to S3
Notes S1 to S8
References
S2
Supplementary Notes 1-3: Cell Strains, Reagents and Materials
Supplementary Note 1: Cell Strains, Reagents and suppliers
All mineralization experiments were conducted in
E. coli
strain DH10B
(
Invitrogen, Carlsbard,
CA).Restriction enzymes, ligase, and Q5 DNA polymerase were purchased from New England
Biolabs (Beverly, NJ). DNA oligos and G-blocks were purchased from Integrated DNA
Technologies (Coralville, IA).
Supplementary Note 2: Plasmid subcloning
Recombinant fusion proteins were produced by standard recombinant DNA technology.
E. coli
strain DH10B was used for all cloning steps and material preparation.
Genes encoding the autotransporter protein along with elastin solubility/stability tags have been
previously cloned by our group into modified pQE-80L plasmids. (
1
)
G-blocks encoding PhoA without its natural leader peptide were ordered with 5’ BamHI and 3’
HindIII sites. G-blocks were digested with BamHI and HindIII and inserted into a similarly
digested pQE-Empty plasmid containing pelB leader peptide described in previous work (
1
) to
make a pelB-PhoA construct that directs PhoA to the periplasm. Using Gibson assembly, the pelB-
PhoA construct was placed under control of the araBAD promoter in pBAD33.
pX-E6 containing six 25-residue elastin-like-peptide repeats reported in previous work (
1
)was
digested with BamHI and XhoI and inserted into a similarly digested pAT-Empty vector to make
pAT-E6. The T5 promoter of the plasmid drives constitutive expression of protein E6-AT.
pX-ST encoding the SpyTag peptide was mutated into pX-N encoding nucleation peptide SN15
(N) (
2
) by PCR. Recursive ligation
(
3
) was used to assemble pX-triblock peptide from pX-N and
pX-E3. pX-triblock was digested with BamHI and XhoI and inserted into a similarly digested
S3
pAT-Empty vector to make pAT-triblock. The T5 promoter of the plasmid drives constitutive
expression of protein triblock-AT.
Supplementary Note 3: Buffer recipe
The 20 mM HEPES buffer used in this work doesn’t contain any PO
4
3-
. The buffer contains 20
mM HEPES, 115 mM NaCl, and 1.2 mM MgCl
2
buffered at pH 7.4. The mineralization buffer
contains calcium glycerophosphate at concentrations ranging from 10 mM to 60 mM.
Name
Backbone/origin/promoter
Purpose
pQE-
Empty
pQE80l/colE1/T5
Empty plasmid for cloning and ampicillin
resistance
pelB-PhoA
pQE80l/colE1/T5
Constitutive expression of PhoA in periplasm
pBAD-
PhoA
pBAD33/p15a/araBAD
Arabinose-inducible expression of PhoA in
periplasm
pAT-E6
pQE80l/colE1/T5
Constitutive expression of E6-AT protein on cell
surface
pAT-
Triblock
pQE80l/colE1/T5
Constitutive expression of triblock-AT surface
displayed nucleation peptide
pAT-
Empty
pQE80l/colE1/T5
Cloning of autotransporter fusion proteins
S4
Table S1: Plasmids used in this study
Table S2: Protein sequences
Protein:
Sequence
pelB-PhoA
MKYLLPTAAAGLLLLAAQPAMAMRGSHHHHHHGSVDRTPEMPVLEN
RAAQGDITAPGGARRLTGDQTAALRDSLSDKPAKNIILLIGDGMGDSEI
TAARNYAEGAGGFFKGIDALPLTGQYTHYALNKKTGKPDYVTDSAAS
ATAWSTGVKTYNGALGVDIHEKDHPTILEMAKAAGLATGNVSTAELQ
DATPAALVAHVTSRKCYGPSATSEKCPGNALEKGGKGSITEQLLNAR
ADVTLGGGAKTFAETATAGEWQGKTLREQAQARGYQLVSDAASLNS
VTEANQQKPLLGLFADGNMPVRWLGPKATYHGNIDKPAVTCTPNPQR
NDSVPTLAQMTDKAIELLSKNEKGFFLQVEGASIDKQDHAANPCGQIG
ETVDLDEAVQRALEFAKKEGNTLVIVTADHAHASQIVAPDTKAPGLT
QALNTKDGAVMVMSYGNSEEDSQEHTGSQLRIAAYGPHAANVVGLT
DQTDLFYTMKAALGLK
pX-E6
pQE80l/colE1/T5
Cloning of pAT-E6
pX-E3
pQE80l/colE1/T5
Cloning of pAT-triblock
pX-ST
pQE80l/colE1/T5
Cloning of pAT-triblock
pX-N
pQE80l/colE1/T5
Cloning of pAT-triblock
S5
E6-AT
MKYLLPTAAAGLLLLAAQPAMAMRGSHHHHHHGSVDVPGAGVPGA
GVPGEGVPGAGVPGAGVPGAGVPGAGVPGEGVPGAGVPGAGVPGAG
VPGAGVPGEGVPGAGVPGAGLDVPGAGVPGAGVPGEGVPGAGVPGA
GVPGAGVPGAGVPGEGVPGAGVPGAGVPGAGVPGAGVPGEGVPGAG
VPGAGLETPTPGPDLNVDNDLRPEAGSYIANLAAANTMFTTRLHERLG
NTYYTDMVTGEQKQTTMWMRHEGGHNKWRDGSGQLKTQSNRYVL
QLGGDVAQWSQNGSDRWHVGVMAGYGNSDSKTISSRTGYRAKASV
NGYSTGLYATWYADDESRNGAYLDSWAQYSWFDNTVKGDDLQSES
YKSKGFTASLEAGYKHKLAEFNGSQGTRNEWYVQPQAQVTWMGVK
ADKHRESNGTLVHSNGDGNVQTRLGVKTWLKSHHKMDDGKSREFQP
FVEVNWLHNSKDFSTSMDGVSVTQDGARNIAEIKTGVEGQLNANLNV
WGNVGVQVADRGYNDTSAMVGIKWQF
Triblock-AT
MKYLLPTAAAGLLLLAAQPAMAMRGSHHHHHHGSVDVPGAGVPGA
GVPGEGVPGAGVPGAGVPGAGVPGAGVPGEGVPGAGVPGAGVPGAG
VPGAGVPGEGVPGAGVPGAGLDDDDEEKFLRRIGRFGLDVPGAGVPG
AGVPGEGVPGAGVPGAGVPGAGVPGAGVPGEGVPGAGVPGAGVPGA
GVPGAGVPGEGVPGAGVPGAGLDDDDEEKFLRRIGRFGLDVPGAGVP
GAGVPGEGVPGAGVPGAGVPGAGVPGAGVPGEGVPGAGVPGAGVPG
AGVPGAGVPGEGVPGAGVPGAGLETPTPGPDLNVDNDLRPEAGSYIA
NLAAANTMFTTRLHERLGNTYYTDMVTGEQKQTTMWMRHEGGHNK
WRDGSGQLKTQSNRYVLQLGGDVAQWSQNGSDRWHVGVMAGYGN
SDSKTISSRTGYRAKASVNGYSTGLYATWYADDESRNGAYLDSWAQ
YSWFDNTVKGDDLQSESYKSKGFTASLEAGYKHKLAEFNGSQGTRNE
WYVQPQAQVTWMGVKADKHRESNGTLVHSNGDGNVQTRLGVKTW
LKSHHKMDDGKSREFQPFVEVNWLHNSKDFSTSMDGVSVTQDGARN
IAEIKTGVEGQLNANLNVWGNVGVQVADRGYNDTSAMVGIKWQF
S6
Fig. S1.
Protein sequence design of autotransporter fusion protein and periplasmic
enzyme. The N-terminal pelB leader peptide directs the translated protein into the
periplasm of
E. coli
and is then cleaved from the protein. The C-terminal
autotransporter sequence is recognized and inserted into the outer membrane of
E.
coli
while the N-terminal part is displayed at the cell surface. PhoA is transported to
the periplasm. (Corresponding color code was applied to
Table S2
.)
Fig. S2.
SRS images of mineralized bacteria in liquid culture in Phosphate, CH3 channel. The
merged image showed core-shell structure in which CH3-containing organics are distributed inside
the cell body while phosphate are forming a shell at the cell periphery. Scale bar: 2 μm.
S7
Fig. S3.
Fluorescence microscopy image of 7 days old (1 day induction with 0.1% arabinose for
PhoA expression) PhoA bacterial film expressing periplasmic PhoA after 24 hrs of mineralization
in 10 mM CGP, stained by the calcium binding dye calcein. The cell interior is not stained by
calcein, consistent with mineralization outside the inner membrane of bacterium. Calcein binds to
calcium cation and becomes fluorescent once binds to calcium, meaning that it binds non-
specifically and cannot distinguish between BCP and other molecules that contains calcium. Scale
bar, 5 μm.
S8
Fig. S4.
A Triblock/PhoA bacterial film after 24 hrs of mineralization in 10 mM CGP, stiff and
self-standing in a 20 mL scintillation vial, already removed from its polycarbonate filter for
imaging and analysis. Diameter of the film is around 2 cm.
S9
Fig. S5.
E. coli
cell liquid culture fully mineralized and stained by calcein (calcium tracker) and
imaged by fluorescence (
a
, calcein) and SRS microscopy (
b
, phosphate channel and
c
, CH
3
channel).
The phosphate channel showed weak SRS signal compared to unstained samples because
phosphate anions were displaced by the calcein-AM dye which chelates calcium ions. Scale bars,
25 μm.
S10
Fig. S6.
a
, A photo and an SRS image of a E6/PhoA film after 120 min mineralization. The edge
was imaged under an SRS microscope. The SRS image was taken in the phosphate channel (ON
– OFF). It is clear that the edge of the film has high intensity of phosphate signal compared to the
interior regions. Scale bars: 1 cm (left, photo), 100 μm (right, SRS image).
b
, SRS spectra of an
E6/PhoA film (after 120-min mineralization) at the edge (blue) and the interior (cyan) regions,
normalized to the highest point in of the spectrum at the edge. The phosphate peak at 961 cm
-1
is
significantly higher at the edge, compared to that from the interior, while the background levels
and shapes remain identical. Number of replicates for each sample: 5. Standard error of mean is
represented by the shaded error bands.
S11
Fig. S7.
Images of PhoA, E6/PhoA and triblock/PhoA cultures. Left bottom tube contains a PhoA
culture. Without surface protein expression, the PhoA culture stays turbid and cells are freely
floating. The middle tube is an E6/PhoA culture. E6 increases intercellular adhesion, causing cells
to aggregate and precipitate, settling to the bottom of the tube. Top right tube is triblock/PhoA.
Many cells are still suspended in the culture, but some precipitate and settle to the tube bottom.
S12
Fig. S8.
SEM images of three types of 7 days old
(1 day induction with 0.1% arabinose for PhoA
expression)
engineered
E. coli
films with different surface protein constructs
after 24 hrs of
mineralization in 10 mM CGP
:
a
, no surface-displayed peptide (PhoA);
b
, with surface-displayed
E6 peptides (E6/PhoA);
c
, with surface-displayed triblock peptides (Triblock/PhoA). Scale bars,
2 μm.
S13
Fig. S9.
Calcium-tracking fluorescence images of three types of 7 days old
(1 day induction with
0.1% arabinose for PhoA expression)
engineered
E. coli
films with different surface protein
constructs
after 24 hrs of mineralization in 10 mM CGP
:
a
, no surface-displayed peptide (PhoA);
b
, with surface-displayed E6 peptides (E6/PhoA);
c
, with surface-displayed triblock peptides
(Triblock/PhoA). Scale bars, 10 μm.
S14
Fig. S10.
Colony-forming units (CFU) per unit area of the three types of films examined in this
work.
Little change in viability was observed during the first hour of mineralization. Relative large
decrease in viability was observed after 90 min of mineralization. Triblock/PhoA has least viability
across all three constructs after 2 h of mineralization.
S15
Supplementary Notes 4: Raman cross section data for quantification of
phosphate/CH (molar ratio)
To get the phosphate/CH ratio in each sample from SRS imaging data, we used the SRS intensity
of both the phosphate and CH channels, as well as the known Raman cross sections of phosphate
P-O bond and C-H bond vibrations (
Table S3
).
푃ℎ표푠푝ℎ푎푡푒
퐶퐻
molar ratio
=
푃ℎ표푠푝ℎ푎푡푒
푃ℎ표푠푝ℎ푎푡푒
퐶퐻
퐶퐻
I
: SRS intensity;
σ
: Raman cross section (
Table S3
)
Table S3: Reported Raman cross sections of phosphate and C-H bond vibrations
(
4
,
5
)
Target
Raman Cross Section (cm
-2
)
Phosphate
8.10E-30
CH
1.00E-29
Supplementary Notes 5: Fluorescence Imaging
The fluorescence images of processed samples with fluorescent labels were obtained with a 25X,
1.05 N.A. water-immersion objective with the Olympus Fluoview system. Single-photon confocal
laser scanning imaging was performed with a 488-nm laser (Coherent OBIS), and emission in the
Calcein-AM channel
(λex/λem:
495/515 nm). The images were visualized and analyzed with Fiji
or Imaris Viewer.
S16
Fig. S11.
a
, Schematic of bulge test device, fabricated as two parts that are separated to load a
sample in the central chamber at the center of the device. When sealed, each external port connects,
via a channel, to a reservoir of fluid that is used to control pressure on the top or bottom face of
the sample respectively (reservoirs not shown). Gray layers are acrylic; green thin layer is cover
slip glass; layers are bonded using epoxy. Horizontal channels are longer than shown.
b
, Schematic
diagram of central chamber. Bacterial film sample is supported by two washer-shaped disks. A
thin O-ring seals this “sandwich” to the top half of the device.
c
, Schematic showing a disk of
bacterial film sample clamped between two washer-shaped supports; when the supports and
specimen are loaded in the central chamber and
p
bottom face
>
p
top face
, the bacterial film bulges
upward (right).
S17
Supplementary Notes 6: Mechanical property obtained by bulge/inflation tests
Mechanical properties of the biofilms were estimated using a bulge/inflation test, a method that
has been used extensively in the characterization of sheet metals, polymers, and some biological
tissues (
6-17
). A millifluidic device was constructed using laser cut acrylic sheets to adapt the
bulge test to the Pa-kPa pressures needed to deform the biofilms. The final device construct
consisted of a sealed central chamber housing the biofilm sample as a 3 mm diameter disk,
sandwiched between two washer-shaped supports (
Fig. S9a, b
). Each face of the sample
was
exposed to a chamber filled with HEPES buffer whose hydrostatic pressure could be independently
controlled. Thus, by applying different hydrostatic pressures to the top and bottom faces of the
biofilm, the film could be “inflated” through the washer aperture into a dome-like shape (
Fig. S9c
).
Hydrostatic pressures were applied across the film by varying the height of HEPES in two separate
reservoirs connected to external
ports in the device. The reservoirs themselves were 60 ml syringes
of known internal diameter (BD Scientific, ID = 26.72 mm, cross sectional area 5.61 cm
2
). Thus,
the addition of 858 μl of HEPES buffer increased the reservoir level by 1.53 mm, which increased
the hydrostatic pressure on the corresponding face of the sample by 15.0 Pa (density assumed
constant at 1 g/ml).
Sample loading
Prior to mechanical testing, a bacterial film with its polycarbonate support was submerged in
HEPES buffer and a 3 mm dia. biopsy punch (Integra Biosciences) was used to gently make a
circular cut through the bacterial film but not the polycarbonate. The sample support disk (copper
TEM disk, 1.5 mm diameter circular aperture, 3.05 mm outer diameter, thickness 25 μm, Ted Pella)
was then slid in between the bacterial film and polycarbonate and used to gently separate the film
from the polycarbonate. Once the edge of the film was freed, the film freely floated away from the
polycarbonate and could be loaded into the device (
Fig. S9b
). The device channels were then filled
with HEPES and the syringe reservoirs attached via luer slip connectors threaded into the device
ports.
Bulge Test and Mechanical Characterization
A millifluidic device was constructed using laser cut acrylic sheets to adapt the bulge test to the
Pa-kPa pressures needed to deform the biofilms. The device consisted of a sealed central chamber
housing the biofilm sample as a 3 mm diameter disk, sandwiched between two washer-shaped
supports. Each face of the sample was exposed to a chamber filled with HEPES buffer whose
hydrostatic pressure could be independently controlled. Thus, by applying different hydrostatic
pressures to the top and bottom faces of the biofilm, the film could be “inflated” through the washer
aperture into a dome-like shape. Hydrostatic pressures were applied across the film by varying the
height of HEPES in two separate reservoirs connected to external ports in the device. The
reservoirs were 60 ml syringes of known internal diameter (BD Scientific, ID = 26.72 mm, cross
sectional area 5.61 cm
2
). Thus, the addition of 858 μl of HEPES buffer increased the reservoir
level by 1.53 mm, which increased the hydrostatic pressure on the corresponding face of the
sample by 15.0 Pa (density assumed constant at 1 g/ml). Prior to mechanical testing, a bacterial
film with its polycarbonate support was submerged in HEPES buffer and a 3 mm diameter biopsy
S18
punch (Integra Biosciences) was used to make a circular cut through the bacterial film but not the
polycarbonate. The sample support disk (copper TEM disk, 1.5 mm diameter circular aperture,
3.05 mm outer diameter, thickness 25 μm, Ted Pella) was then inserted between the bacterial film
and polycarbonate and used to gently separate the film from the polycarbonate. Once the edge of
the film was freed, the film freely floated away from the polycarbonate and could be loaded into
the device. The device channels were then filled with HEPES and the syringe reservoirs attached
via luer slip connectors threaded into the device ports. A syringe pump (KD Scientific) was used
to add HEPES buffer to the reservoir connected to the bottom face of the biofilm at a flow rate of
70 ml/min with a final volume of 55 ml. Biofilm deformation was imaged using optical coherence
tomography (OCT, Thorlabs, GAN210 base unit: 930 nm central wavelength, 6 μm axial
resolution. OCTP-900 scan head, OCT-LK3-BB scan lens: 36 mm FL, 8 μm lateral resolution).
A-Scan/Line Rate was 36 kHz (acquisition time = 19 ms).
Operation and analysis
A syringe pump (KD Scientific) was used to add HEPES buffer to the reservoir connected to the
bottom face of the biofilm at a flow rate of 70 ml/min with a final volume of 55 ml. The biofilm
deformation was imaged using optical coherence tomography (OCT, Thorlabs, GAN210 base unit:
930 nm central wavelength, 6 μm axial resolution. OCTP-900 scan head, OCT-LK3-BB scan lens:
36 mm FL, 8 μm lateral resolution). A-Scan/Line Rate was 36 kHz (acquisition time = 19 ms).
Analysis protocols: Biofilm thickness ranged from 55-120 μm with a majority of films under 80
μm in thickness. Thus, the ratio of aperture diameter to film thickness ranged from 12.5 to 27.2.
Flexural stiffness was neglected and only membrane stresses and strains were considered. Analysis
of 2D images further required the spherical cap assumption: that there is equibiaxial stress and
strain everywhere in the film and that the bulge radius of curvature is the same in both radial and
circumferential directions – these assumptions are generally only satisfied at the center of the film
and fail near the clamped edge where the circumferential strain vanishes.
Analysis of 2D images: 2D OCT datasets were exported as tiff files and cropped using ImageJ.
The resulting images were processed with in-house MATLAB scripts: the images were binarized;
the top and bottom surfaces of the film were automatically detected and fit to fourth degree
polynomials which were then used to estimate arc lengths of the top and bottom surfaces of the
film. The arc lengths were used to calculate true strain averaged over the top and bottom surfaces
of the biofilm.
True stress was calculated with the equation for stress in a thin-walled spherical pressure vessel:
=
푃푅
2푡
Where
is film stress, is the applied pressure, is bulge radius of curvature, and is
instantaneous film thickness. Further,
where is radius of aperture of the support
=
2
+
2
disk and is deflected height of the center of the film, directly measurable by OCT. Young’s
modulus was estimated using the initial linear regime of each stress-strain trace.
S19
Supplementary Notes 7: Mineralization Protocols
Mineralization of Planktonic Bacterial Cultures
Overnight
E. coli
cultures were diluted 100-fold and grown to OD
600
of 0.4–0.6 prior to induction
with 0.1% L-arabinose. Expression was allowed to proceed for 90 min, after which the culture was
centrifuged and resuspended in 20 mM HEPES buffer (pH 7.4) containing 10 mM calcium
glycerophosphate. The detailed buffer recipe is documented in Supplementary Note 3.
Mineralization was allowed to proceed for at least 1 h under shaking at 250 rpm until cells
precipitated as white clusters. Clusters were resuspended and a 3 μL aliquot of resuspended
mineralized cells was transferred to a plastic chamber (120 μm depth, Thermo-Fisher) sandwiched
between a glass slide and a cover slip for Raman microscopy.
Full Mineralization of Bacterial Films
Bacterial films were prepared as described in the above sections and transferred on their
polycarbonate filters to a 6-well plate (Falcon) with each well filled with 5 mL of 20 mM HEPES
buffer (pH 7.4) containing 10 mM calcium glycerophosphate. Four 3 mm diameter glass beads
were placed at the edge of each polycarbonate filter to keep the films flat during the mineralization
process. To achieve full mineralization, films remained in mineralization buffer for 24 h.
Microtomy of Fully-Mineralized Bacterial Films
Fully-mineralized films were removed from the mineralization buffer, washed with 5 mL of 20
mM HEPES buffer for 5 min and then cut with a 6 mm diameter biopsy punch (Miltex). The
circular section of film was embedded in Tissue-Tek® resin (Sakura) and frozen at - 20 ºC
overnight. The frozen piece was microtomed at – 20 ºC with each section of 50 μm thickness.
Microtomed sections were placed either on glass slides (for Raman microscopy) or on Al stubs
(for SEM imaging).
Supplementary Notes 8: Sample Mounting and Imaging for SRS
Cell or biofilm samples were kept in HEPES buffer for imaging. Grace Bio-Labs SecureSeal™
spacers with appropriate opening sizes were used as spacers between microscope slides (1 mm,
VWR) and coverslips (12 mm, #1.5, Fisher). Confocal images were obtained by the Olympus
FluoView™ FV3000 confocal microscope with the SRS setup described above.
S20
S21
Fig. S12.
a
, True stress vs. true strain curves of all 7 days old PhoA films mineralized at different
time points. Number of replicates for each time point: 20 min, n = 3; 30 min, n = 3; 40 min, n =3,
60 min n = 3; 90 min, n = 3; 120 min, n = 2.
b
, True stress vs. true strain curves of all 7 days old
E6/PhoA films mineralized at different time points. Number of replicates for each time point: 0
min, n = 3; 20 min, n = 3; 30 min, n =3, 40 min n = 2; 60 min, n = 1; 90 min, n = 3.
c
, True stress
vs. true strain curves of all 7 days old Triblock/PhoA films mineralized at different time points.
Number of replicates for each time point: 20 min, n = 3; 30 min, n = 3; 40 min, n =4, 60 min n =
4; 90 min, n = 3; 120 min, n = 4.
S22
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