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Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion

Published: February 10, 2023 doi: 10.3791/64382


This article provides a straightforward and clear protocol to label Salmonella secreted effectors using genetic code expansion (GCE) site-specifically and image the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)


Type three secretion systems (T3SSs) enable gram-negative bacteria to inject a battery of effector proteins directly into the cytosol of eukaryotic host cells. Upon entry, the injected effector proteins cooperatively modulate eukaryotic signaling pathways and reprogram cellular functions, enabling bacterial entry and survival. Monitoring and localizing these secreted effector proteins in the context of infections provides a footprint for defining the dynamic interface of host-pathogen interactions. However, labeling and imaging bacterial proteins in host cells without disrupting their structure/function is technically challenging.

Constructing fluorescent fusion proteins does not resolve this problem, because the fusion proteins jam the secretory apparatus and thus are not secreted. To overcome these obstacles, we recently employed a method for site-specific fluorescent labeling of bacterial secreted effectors, as well as other difficult-to-label proteins, using genetic code expansion (GCE). This paper provides a complete step-by-step protocol to label Salmonella secreted effectors using GCE site-specifically, followed by directions for imaging the subcellular localization of secreted proteins in HeLa cells using direct stochastic optical reconstruction microscopy (dSTORM)

Recent findings suggest that the incorporation of non-canonical amino acids (ncAAs) via GCE, followed by bio-orthogonal labeling with tetrazine-containing dyes, is a viable technique for selective labeling and visualization of bacterial secreted proteins and subsequent image analysis in the host. The goal of this article is to provide a straightforward and clear protocol that can be employed by investigators interested in conducting super-resolution imaging using GCE to study various biological processes in bacteria and viruses, as well as host-pathogen interactions.


Bacterial infections have long been regarded as a serious hazard to human health. Pathogens use highly evolved, extremely powerful, and intricate defense systems, as well as a variety of bacterial virulence factors (referred to as effector proteins) to evade host immune responses and establish infections1,2. However, the molecular mechanisms underlying these systems and the role of individual effector proteins are still largely unknown due to the dearth of suitable approaches for directly following the crucial protein components and effectors in host cells during pathogenesis.

One typical example is Salmonella enterica serovar Typhimurium, which causes acute gastroenteritis. Salmonella Typhimurium uses type three secretion systems (T3SS) to inject a variety of effector proteins directly into host cells. As soon as Salmonella enters the host cell, it resides in an acidic membrane-bound compartment, termed the Salmonella-containing vacuole (SCV)3,4. The acid pH of the SCV activates the Salmonella pathogenicity island 2 (SPI-2)-encoded T3SS and translocates a volley of 20 or more effector proteins across the vacuolar membrane into the host cytosol5,6,7,8. Inside the host, these complex cocktails of effector proteins coordinately manipulate host cell signaling pathways, resulting in the formation of highly dynamic, complex tubular membrane structures extended from the SCV along microtubules, termed Salmonella-induced filaments (SIFs), that enable Salmonella to survive and replicate within the host cells9,10,11.

Methods to visualize, track, and monitor bacterial effector localizations, and examine their trafficking and interactions inside host cells, provides critical insight into the mechanisms underpinning bacterial pathogenesis. Labeling and localization of Salmonella secreted T3SS effector proteins inside host cells has proven to be a technological challenge12,13; Nonetheless, the development of genetically-encoded fluorescent proteins has transformed our ability to study and visualize proteins within living systems. However, the size of fluorescent proteins (~25-30 kDa)15 is often comparable to or even greater than that of the protein of interest (POI; e.g., 13.65 kDa for SsaP, 37.4 kDa for SifA). In fact, fluorescent protein labeling of effectors often blocks the secretion of the labeled effector and jams the T3SS14.

Furthermore, fluorescent proteins are less stable and emit a low number of photons before photobleaching, limiting their use in super-resolution microscopic techniques16,17,18, particularly in photoactivation localization microscopy (PALM), STORM, and stimulated emission depletion (STED) microscopy. While the photophysical properties of organic fluorescent dyes are superior to those of fluorescent proteins, methods/techniques such as CLIP/SNAP19,20, Split-GFP21, ReAsH/FlAsH22,23, and HA-Tags24,25 require an additional protein or peptide appendage that may impair the structure-function of the effector protein of interest by interfering with post-translational modification or trafficking. An alternative method that minimizes necessary protein modification involves the incorporation of ncAAs into a POI during translation through GCE. The ncAAs are either fluorescent or can be made fluorescent via click chemistry12,13,26,27,28.

Using GCE, ncAAs with tiny, functional, bio-orthogonal groups (such as an azide, cyclopropene, or cyclooctyne group) can be introduced at nearly any location in a target protein. In this strategy, a native codon is swapped with a rare codon such as an amber (TAG) stop codon at a specified position in the gene of the POI. The modified protein is subsequently expressed in cells alongside an orthogonal aminoacyl-tRNA synthetase/tRNA pair. The tRNA synthetase active site is designed to receive only one particular ncAA, which is then covalently attached to the 3'-end of the tRNA that recognizes the amber codon. The ncAA is simply introduced into the growth medium, but it must be taken up by the cell and reach the cytosol where the aminoacyl-tRNA synthetase (aaRS) can link it to the orthogonal tRNA; it is then incorporated into the POI at the specified location (see Figure 1)12. Thus, GCE enables site-specific incorporation of a plethora of bio-orthogonal reactive groups such as ketone, azide, alkyne, cyclooctyne, transcyclooctene, tetrazine, norbonene, α, β-unsaturated amide, and bicyclo [6.1.0]-nonyne into a POI, potentially overcoming the limitations of conventional protein labeling methods12,26,27,28.

Recent emerging trends in super-resolution imaging techniques have opened up new avenues to investigate biological structures at the molecular level. In particular, STORM, a single-molecule, localization-based, super-resolution technique, has become an invaluable tool to visualize cellular structures down to ~20-30 nm and is able to investigate biological processes one molecule at a time, thereby discovering the roles of intracellular molecules that are yet unknown in traditional ensemble-averaged studies13. Single-molecule and super-resolution techniques require a small tag with bright, photostable organic fluorophores for the best resolution. We recently demonstrated that GCE can be used for incorporating suitable probes for super-resolution imaging12.

Two of the best choices for protein labeling in cells are bicyclo [6.1.0] nonynelysine (BCN) and trans-cyclooctene-lysine (TCO; shown in Figure 1), which may be genetically encoded using a variant of the tRNA/synthetase pair (here termed tRNAPyl/PylRSAF), where Pyl represents pyrrolysine, and AF represents a rationally designed double mutant (Y306A, Y384F) derived from Methanosarcina mazei that naturally encodes pyrrolysine12,29,30,31. Through the strain-promoted inverse electron-demand Diels-Alder cycloaddition (SPIEDAC) reaction, these amino acids react chemoselectively with tetrazine conjugates (Figure 1)12,30,31. Such cycloaddition reactions are exceptionally fast and compatible with living cells; they may also be fluorogenic, if an appropriate fluorophore is functionalized with the tetrazine moiety12,26,32. This paper presents an optimized protocol for monitoring the dynamics of bacterial effectors delivered into host cells using GCE, followed by subcellular localization of secreted proteins in HeLa cells using dSTORM. The results indicate that incorporation of an ncAA via GCE, followed by a click reaction with fluorogenic tetrazine-bearing dyes, represents a versatile method for selective labeling, visualization of secreted proteins, and subsequent sub-cellular localization in the host. All the components and procedures detailed here, however, can be adjusted or substituted so that the GCE system can be adapted to investigate other biological questions.

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1. Plasmid construction

  1. Clone the gene expressing the POI into an expression plasmid (e.g., pET28a-sseJ10TAG) that expresses the POI in E. coli BL21 (DE3). This step facilitates in determining that the mutants are functional. 
  2. For the visualization of Salmonella secreted effectors in host cells, construct an expression plasmid (pWSK29-sseJ-HA) that expresses the target POI SseJ under the control of its native promoter, as described in previous reports7,12,24. Using site-directed mutagenesis, replace the codon of Phenylalanine-10 with an amber codon (TAG) in SseJ (pWSK29-sseJ10TAG-HA), ensuring the AAT-TAG-ACT motif33,34.
  3. In addition to the above plasmids, for the genetic incorporation of trans-cyclooct-2-en-L-lysine (TCO*A) into a POI, use another expression plasmid (pEVOL-PylRS-AF)31 that contains the evolved tRNA/synthetase pair (tRNAPyl/PylRSAF ) genes encoded in the plasmid pEVOL.
    NOTE: Detailed descriptions of the plasmid and the cloning protocols are described in the previous report30,31.
  4. Use commercially available plasmid preparation kits to obtain high-quality plasmid DNA. For purified DNA, the optimal 260/280 ratio is ~1.8. Check the DNA purity using 1% agarose gel electrophoresis, if necessary.

2. Bacterial culture preparation

  1. Obtaining double transformants to test that the mutant is functional in E. coli
    1. Take BL21 competent cells out of -80 °C and thaw on ice for approximately 20–30 min.
    2. Mix 1–5 μL of each plasmid (~50 ng of pEVOL-PylRS-AF and pET28a-sseJ10TAG) with 200 μL of chemically competent BL21 cells in a 1.5 mL microcentrifuge tube. Mix the competent cells and plasmid mixture gently; incubate for 30 min on ice.
    3. Heat-shock the microcentrifuge tube in a 42 °C water bath for 45 s. Put the tubes back on ice for 2 min.
    4. Add 1 mL of warm LB medium to the microcentrifuge tube and quickly transfer the mixture into a 15 mL conical tube. Incubate the cells at 37 °C in a shaker at 250 rpm for 1 h. Plate some portion or all the transformed cells on LB agar plates containing appropriate antibiotics. Incubate the plates overnight at 37 °C.
      NOTE: In this protocol, 35 µg/mL chloramphenicol and 50 µg/mL kanamycin were used for selecting pEVOL-PylRS-AF and pET28a-sseJ10TAG, respectively. In the transformation step, evaluate the successful transformation using negative and positive controls.
  2. Transfer the mutant to Salmonella for the visualization of Salmonella secreted effectors in host cells:
    1. Add 2–3 µL each of pEVOL-PylRS-AF and pWSK29-sseJ10TAG to 40–60 µL of electrocompetent Salmonella Typhimurium strain 14028s in a chilled microcentrifuge tube. Mix the mixture gently with a pipette.
    2. Transfer the electroporation mixture into a chilled (0.2 cm electrode gap) cuvette; set the electroporation settings to 2.5 kV, 200 Ω, 25 µF. Place the cuvette inside the electroporation chamber. Make sure that the chamber electrode makes firm contact with the micro pulser cuvette.
    3. Press and hold the pulse button until it beeps.
    4. Immediately add 1 mL of warm LB medium into the cuvette. Transfer the entire contents into a 15 mL conical tube. Incubate the cells at 37 °C with shaking at 250 rpm for 1 h.
    5. Plate some portion or all of the electroporation mixture of Salmonella on an LB agar plate containing appropriate antibiotics. Incubate the plates overnight at 37 °C.
      NOTE: In this protocol, 35µg/mL chloramphenicol and 100 µg/mL ampicillin were used for the selection of pEVOL-PylRS-AFand pWSK29-sseJ10TAG, respectively.
  3. Culture preparation
    1. Inoculate a single colony of Salmonella or E. coli in 5 mL of LB medium containing appropriate antibiotics. Grow overnight at 37 °C, shaking at 250 rpm.

3. Expression and fluorescent labeling of ncAA-bearing proteins

  1. Expression of ncAA-bearing proteins in E. coli
    1. Transfer 100 µL of primary culture into 5 mL of LB medium (1:50 dilution) containing 35 µg/mL chloramphenicol and 50 µg/mL kanamycin, followed by incubation at 37 °C with shaking at 250 rpm until OD600 reaches 0.4–0.6.
    2. Prepare 1 mM TCO*A stock solution (10 mL; Table 1).
    3. When the cultures reach OD600 = 0.4–0.6, replace the LB media with fresh LB containing 1 mM TCO*A, 35 µg/mL chloramphenicol, and 50 µg/mL kanamycin.
      NOTE: Alternatively, TCO*A stock solution can be prepared as described in step 5.4.2.
    4. Induce protein expression in the presence of 1 mM IPTG, 0.2% arabinose and shake overnight at 34 °C, 250 rpm. Harvest the bacterial cells by centrifugation for 5 min at 11,000 × g. Remove the supernatant and freeze the pellet at -20 °C until further use.
    5. For a control experiment, repeat the same experiment but express the ncAA-bearing proteins in the absence of the ncAA. For in vitro fluorescent labeling of ncAA-bearing proteins in E. coli, follow the detailed procedure described in step 3.3.
  2. Expression of ncAA-bearing proteins in Salmonella
    1. Transfer 100 µL of primary culture into 5 mL of LB medium (1:50 dilution) containing 35 µg/mL chloramphenicol and 100 µg/mL ampicillin, followed by incubation at 37 °C with shaking at 250 rpm until OD600 reaches 0.6.
    2. Prepare modified N-minimal medium (MgM, pH 5.6) as described in Table 1.
    3. Replace the LB medium with modified N-minimal medium (MgM) supplemented with 1 mM TCO*A (Table 1). Grow the bacteria at 34 °C for 30 min. Then, add 0.2% arabinose, 25 mg/mL chloramphenicol, and 100 mg/mL ampicillin, and grow the cells for another 6 h, shaking at 250 rpm.
    4. After 6 h, wash the bacteria 4x over an interval of 30 min, with fresh MgM (pH 5.6) media (Table 1) without ncAA. In the washing steps, use 972 × g for 15 min at room temperature.
    5. Centrifuge the bacteria, resuspend in 1x PBS buffer, and incubate for 1 h at 4 °C in the dark to remove excess ncAAs. After 1 h, centrifuge the bacteria at 3,000 × g, 4 °C for 15 min and store for further use.
    6. For a control experiment, repeat the same experiment by expressing ncAA-bearing proteins in the absence of ncAA.
  3. In vitro fluorescence labeling of ncAA-bearing proteins in Salmonella Typhimurium
    1. Resuspend Salmonella cells expressing SseJ-F10TCO-HA in the absence or presence of TCO*A (from step 3.2) in 1x PBS. Adjust the OD600 to 4 in PBS. Incubate the cells with 20 µM Janelia Fluor 646-tetrazine (JF646-Tz) or 20 µM BDP-Fl-tetrazine (5 mM stock solutions in DMSO) at 37 °C in the dark and shake for 1–2 h at 250 rpm.
    2. Pellet the cells, wash 3–4x with PBS containing 5% DMSO and 0.2% Pluronic F-127, resuspend in PBS containing 5% DMSO, and incubate overnight at 4 °C in the dark. Wash 2x again with 1x PBS. In the washing steps, use 972 × g, for 15 min at room temperature.
    3. Image the cells immediately using a confocal microscope or fix them with 1.5% paraformaldehyde (PFA) in PBS for 30–45 min at room temperature in the dark.
    4. Wash the fixed cells 2x with PBS and finally in 50 mM NH4Cl in PBS for 15 min to remove excess PFA. Resuspend the cells in PBS and store at 4 °C for no more than 3–4 days. Image the bacteria using confocal microscopy as described in section 6.

4. Biochemical characterization of ncAA-bearing proteins

  1. Cell lysis
    1. Resuspend the cells from section 3.1 in lysis buffer (Table 1) and incubate at room temperature for more than 30 min. Chill the mixture on ice for 15 min.
      NOTE: Keep the ratio of lysis buffer to cell weight used to 1:10.
    2. Sonicate the resuspended cells while still on ice. Pulse for 30 s at setting 7 at least 6x, with 1 min gaps, using a sonicator (see the Table of Materials).
    3. Spin down the cell lysate at 20,500 × g, 4 °C for 15 min (maintaining 4 °C is critical). Transfer the supernatant to a fresh tube and discard the pellet.
  2. SDS-PAGE analysis
    1. Prepare 5x SDS loading dye (Table 1). Add 5 µL of SDS gel loading buffer to 13 µL of cell lysate. Denature the proteins with sodium dodecyl sulfate (SDS) sample buffer in a 2 mL tube at 95 °C for 10 min, centrifuge the mixture at 2,000 × g for 50 s, and load onto a 12% SDS polyacrylamide gel.
    2. Assemble the gel cassette, place it in a gel electrophoresis apparatus, and connect the electrophoresis apparatus to an electric power supply. Pour the running buffer, load the molecular weight markers and protein samples, and run the gel at 100 V, until the bromophenol reaches the bottom of the resolving gel.
      NOTE: Begin the analysis immediately, as lysates stored at -20 °C tend to lose quality after each freeze-thaw cycle.
    3. Stain the gel with Coomassie blue protein stain.

5. Bio-orthogonal labeling of  Salmonella secreted effector SseJ-F10TCO-HA in HeLa cells

  1. Culture and maintain HeLa cells at 37 °C, 5% CO2, and 95% humidity in a high-glucose (4.5 g/L) Dulbecco's modified eagle medium (DMEM), supplemented with 10% (v/v) FBS in addition to Penicillin-Streptomycin (1x).
  2. Culture bacteria 1 day before infection. Inoculate a single colony of Salmonella 14028s harboring pEVOL-PylRS-AF and pWSK29-sseJ10TAG in 5 mL of antibiotic-containing standard LB broth overnight at 37 °C, shaking at 250 rpm.
  3. Seed HeLa cells 1 day before infection. Take a tissue culture flask (T-75) containing HeLa cells at ~80% confluency, as determined by microscopic examination of the cell density. Wash the cells with warm PBS. Detach the cells with 3 mL of warm 0.25% trypsin/EDTA for 5 min at 37 °C, 5% CO2.
    1. Quench the trypsin using 2 mL of DMEM (+ 10% FBS). Transfer the entire contents of the flask into a 15 mL tube after resuspending the cells. Spin down the cells at 1,000 × g for 5 min. Take the supernatant out of the tube. Resuspend the HeLa cell pellet in prewarmed growth medium.
    2. Use a hemocytometer to examine the suspension and count the cells present. Prepare a diluted cell stock at 1 × 105 cells/mL. Seed 0.5 × 105 HeLa cells per well in 500 µL of DMEM + 10% FBS growth medium in an eight-well chamber slide. Keep the chamber slide in an incubator at 37 °C, 5% CO2, 95% humidity for 24 h.
  4. Bacterial infection
    1. Subculture Salmonella 14028s harboring pEVOL-PylRS-AF and pWSK29-sseJ10TAG, by diluting 100 µL of overnight bacterial culture (from step 5.2) into 3 mL of LB medium (1:30 dilution) containing 35 µg/mL chloramphenicol and 100 µg/mL ampicillin, followed by incubation at 37 °C with shaking at 250 rpm for 5–7 h.
      NOTE: During this phase, the cultures reach the late exponential phase, and the bacteria are highly invasive.
    2. Prepare 100 mM TCO*A stock solution and complete media (Table 1).
    3. To initiate the HeLa cell infection, take out the HeLa cells from the incubator and wash the cells with prewarmed DPBS before adding 500 μL of fresh DMEM (+10% FBS) to each well. Place the chamber slide back in the CO2 incubator until the infection begins.
    4. Following 5–7 h of incubation, dilute the Salmonella culture to OD600 = 0.2 in 1 mL of DMEM growth medium (~3 × 108 cfu/mL). Add the requisite amounts of the Salmonella inoculum in each well of the chamber slide so that the multiplicity of infection (MOI) is 100.
    5. Incubate the infected cells in a CO2 incubator for 30 min. Wash the cells 3x with prewarmed DPBS to remove extracellular Salmonella. Set this time point to 0 h post infection. Add 500 µL of complete medium (Table 1) containing 100 μg/mL gentamicin for 1 h. After 1 h, wash the cells 3x with DPBS. Add 500 μL of the complete medium (from step 5.4.2; Table 1) supplemented with 0.2% arabinose, 10 μg/mL gentamicin to each well of the chamber slide.
    6. In a control experiment, perform similar infections of HeLa cells without TCO*A.
    7. Incubate the chamber slide for 10 h in the CO2 incubator.
  5. Click chemistry-based labeling in live cells
    1. Proceed to label the bacteria-infected cells. Prewarm the complete medium without TCO*A (Table 1).
    2. After 10 h post infection, replace the complete medium with fresh complete medium without TCO*A and wash the HeLa cells 4x over an interval of 30 min each with prewarmed DPBS, followed by fresh complete medium (without TCO*A).
    3. Prepare a 0.5 mM dye-tetrazine stock solution in DMSO.
      NOTE: For labeling Salmonella secreted effectors in host cells, two protocols are presented. For protocol 1, prepare the dye mixture by diluting JF646-Tz stock in 1x PBS containing 1% casein sodium salt from bovine milk so that the final working solution is 2 µM. For protocol 2, prepare warm complete medium (without FBS and TCO*A) and add 2 µM JF646-Tz. Make this dye mixture fresh for each labeling session. Work in the dark if possible (dim the lights in the hood and/or the room).
    4. After 12 h post infection, aspirate off the medium from the cells. Wash the cell with prewarmed DPBS 2x or 3x. In one group of wells, add 500 μL of the dye solution mixture described in protocol 1, and in another group of wells of the same chamber slide, add 500 μL of the dye solution mixture described in protocol 2 mentioned in step 5.5.3 and the note after. Place the chamber slides back into the CO2 incubator for 1.5–2 h.
    5. After 13.5–14 h post infection, rinse the HeLa cells 2x with prewarmed DPBS. Add 500 μL of fresh DMEM (supplemented with FBS). Place the chamber slide back in the incubator for 30 min. Wash the cells with prewarmed DPBS followed by fresh DMEM 4x over an interval of 30 min.
    6. At 16 h post infection, fix the HeLa cells with PFA by adding 200 μL of 4% PFA in each well and incubating for 10 min at room temperature in the dark. Aspirate off the PFA, rinse 3x with PBS, and store the cells in PBS at 4 °C in the dark.
      ​NOTE: PFA is a highly toxic chemical. Avoid inhalation, as well as skin and eye contact. Wear protective equipment when handling. To avoid the labeled sample from being exposed to light, turn off the light in the cell culture hood.
  6. Immunostaining of HA-tagged proteins
    1. Aspirate off the PBS and rinse once in blocking solution (Table 1).
    2. Incubate the fixed HeLa cells with a PBS-based primary antibody solution (Table 1) for 1 h at room temperature in the dark. Use a rabbit anti-HA primary antibody to stain secreted SseJ (1:500 dilution).
    3. After 1 h, gently wash the cells 2x with 0.5 mL of 0.1% Tween-20/1x PBS. During the washes with Tween/PBS solution, incubate the cells 3x with 0.5 mL of 0.1% Tween20/1x PBS for 5 min.
    4. Dilute secondary antibody donkey anti-rabbit 555 1:500 in secondary antibody solution and incubate for 1 h at room temperature in the dark.
    5. After 1 h, gently wash the cells 2x with 0.5 mL of 0.1% Tween-20/1x PBS and incubate 2x with 0.5 mL of 0.1% Tween20/1x PBS for 5 min.
    6. Wash the cells 3x with 0.5 mL of 1x PBS and store them at 4 °C in the dark.
      NOTE: Fixed cells can be stored for a couple of weeks in PBS at 4 °C. Immunostaining should be performed at the last minute.

6. Confocal imaging

  1. Use a confocal microscope, such as spinning disc (SD) or STED microscope.
    1. Adjust the image acquisition settings, such as scan mode (XYZ), scanning speed (400 Hz), resolution (512 x 512), magnification (100x), and Z-stacking.
    2. Use the correct excitation/emission filters for DAPI, BDP-Tz, and JF646 tetrazine.
      ​NOTE: For this protocol, confocal imaging was performed on a STED microscope system equipped with a pulsed white light laser, allowing the selection of excitation in the range of 470–670 nm and a UV 405 nm diode laser for excitation at 405 nm, 488 nm, and 647 nm. The appropriate emission ranges were set up using the built-in acousto-optical beam splitter in combination with a prism-based tunable multiband spectral detection of the confocal system.
    3. Acquire the images using a 100×/1.4 oil immersion objective.

7. Super-resolution (dSTORM) imaging

  1. Turn on the microscope 3 h before imaging to permit thermal equilibrium (drift is more likely to occur if imaging begins before this). Cool the camera to -70 °C.
  2. Meanwhile, prepare fresh GLOX-BME imaging buffer (Table 1).
  3. Bring the cells to the microscope. Place the imaging chamber on the microscope stage in the sample holder. In the holder, make sure the sample is flat and secure. Change the medium in the well with 0.4 mL of the freshly made GLOX-BME imaging buffer.
  4. Set the correct laser line and filter sets. Decrease the laser power to ~1 mW to identify a HeLa cell of interest. Adjust the focal plane and laser beam angle while illuminating the sample with low 647 nm laser densities (in this case, a steeply inclined angle [HILO] is suggested, since HILO raises the signal-to-background [SBR]). Adjust the HILO illumination angle.
    NOTE: In this protocol, super-resolution imaging of SseJ was acquired in the Alexa Fluor 647 channel using an inverted, epifluorescence microscope (see the Table of Materials), equipped with a TIRF objective (100x Apo TIRF oil immersion objective, NA 1.49). Fluorescence was detected with an EMCCD camera, which was controlled through μManager.
  5. Set the preamplifier gain to 3 and activate the frame transfer in μManager. Set EM-gain to 200 for higher sensitivity in dSTORM measurements, as described in a previous report35.
  6. Prior to dSTORM imaging, capture a reference diffraction-limited image of the target structure. Switch the fluorophores to the dark state by turning on the laser to its maximum power.
    NOTE: Individual, rapidly blinking molecules of JF646 fluorophores were observed when JF646 fluorophores were transformed to a predominantly dark state by continuous illumination of 647 nm excitation light, as described previously36.
  7. Adjust the laser strength to a suitable level where blinking events are separated in space and time, set the exposure time to 30 ms, and begin the acquisition. Acquire 10,000–30,000 frames.
    NOTE: Changes in laser strength should be made to optimize blinking. Consider increasing laser intensity if there are too many events (blinking particles that overlap) detected. The ideal length depends on the quality of the sample, but ~10,000 frames should show discernible features.

8. Image reconstruction of dSTORM

  1. Use appropriate software for image analysis.
    NOTE: ThunderSTORM, one of many open-source image reconstruction programs available, is briefly described below.
  2. Open ImageJ and import the raw data. Open the ThunderSTORM plugin and configure the camera setting corresponding to the device.
  3. Go to Run Analysis and set the appropriate settings, such as image filtering (difference of averaging filters), localization methods (local maximum), and sub-pixel localization of molecules (integrated Gaussian PSF). Click OK to start image reconstruction.
    NOTE: If necessary, detailed instructions for setting up the parameters in ThunderSTORM have already been described37.

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Representative Results

This protocol paper describes a GCE-based method for site-specific fluorescent labeling and visualization of Salmonella secreted effectors, as depicted in Figure 1. Chemical structure of the ncAA bearing trans-cyclooctene bioorthogonal group (TCO) and the fluorescent dye are shown in Figure 1A. SseJ labeling was achieved by genetic incorporation of bioorthogonal ncAAs at an amber stop codon (see Figure 1B) using an orthogonal aminoacyl-tRNA synthetase/tRNA pairs via GCE technology. Treatment of TCO-labeled effectors with the tetrazine containing a fluorophore (JF646-Tz or BDP-Tz) provided an alternate protein labeling strategy that enabled the observation of translocated Salmonella secreted effectors many hours after infection of the host (Figure 1B,C). We began by identifying possible amino acid residues and selected position 10 of SseJ for ncAA inclusion based on surface accessibility and existing knowledge of SseJ functional residues. Prior structural data and surface exposure projections can aid in the selection of specific locations.

The placement of the amber codon within the gene encoding the POI affects the efficacy of TCO incorporation. As a result, a series of mutants with amber codons in various positions of the gene of the POI should be created and analyzed for optimum integration efficiency. Selection of the amber codon position is empirical. In general, the altered residues should be non-essential for POI function, devoid of post-translational modifications, and accessible for fluorescent probes. The expression plasmid should be a low copy number so that it mimics the native copy number of the POI. The codon context affects how effectively an ncAA is incorporated by an orthogonal tRNA/aminoacyl-tRNA synthetase system. While AAT-TAG-ACT is the preferred context of the orthogonal tRNA, the position of Phenylalanine-10 of sseJ was changed to TAG, ensuring the AAT-TAG-ACT context for enabling the most effective incorporation of the ncAA. First, we tested whether the mutants were functional in E. coli using a high copy number plasmid to express sseJ. As evidenced by SDS-PAGE, SseJ-F10TCO-HA mutant expression was dependent on the presence of TCO*A and the corresponding optimal tRNA/aaRS (Figure 2A). SseJ-F10FCO-HA was labeled with JF646-Tz using SPIEDAC click chemistry in E. coli. The selective fluorescent labeling of SseJ-F10TCO-HA in E. coli in vitro was confirmed by fluorescence microscopy analysis (Figure 2B). Using GCE, TCO*A was thus site-specifically incorporated at an amber codon in sseJ. Attempts to directly identify genomic off-target incorporation of ncAAs using tRNAPyl/PylRSAF system produced no evidence of their existence. However, we recommend using in-gel fluorescence, Western blot, fluorescent fusion protein, in vitro fluorescence labeling, and other methods to assess the efficiency, specificity, degree of off-target incorporation, and toxicity of the pEVOL plasmid, as documented in previously reported papers cited herein12,29.

Once we established that the suppressed TAG in sseJ was functional, we cloned sseJ-F10TCO-HA along with its native promoter into the low copy number plasmid pWSK29, ensuring an AAT-TAG-ACT motif for fluorescent labeling and visualization. In vitro selective labeling of SseJ-F10TCO-HA in Salmonella cells was confirmed using fluorescence microscopy (Figure 3). We then infected HeLa cells with the wild-type Salmonella strain complemented with psseJ-HA or psseJ10TAG-HA, in the absence or presence of TCO*A to determine whether the TCO-incorporated SseJ could be translocated into host cells. Infected HeLa cells were fixed with PFA and immunostained with anti-HA antibody 16 h post infection to highlight the SIFs decorated by SseJ. As shown in Figure 4C, SseJ-F10TCO-HA was obviously secreted, SseJ-dependent SIFs were formed, and they looked similar to those observed in wild-type infected cells (Figure 4A). However, SseJ-dependent SIFs were not observed in the absence of TCO*A (Figure 4B). These observations demonstrated that the expression of sseJ-F10TCO-HA using GCE rescued SseJ-dependent SIFs. Additionally, the results also indicated that SseJ-F10TCO-HA was a fully functional virulence factor for Salmonella.

After confirming that the GCE-labeled SseJ was functional and secreted, we used the SPIEDAC reaction to label SseJ-F10TCO-HA with JF646-Tz in HeLa cells. To do so, we infected HeLa cells with wild-type Salmonella complemented with psseJ-F10TCO-HA, in the presence of TCO*A. After labeling with JF646-Tz using two alternative protocols described in protocol section 5, cells were washed extensively to remove excess dye, fixed with PFA, and immunostained with an anti-HA antibody to visualize the secreted SseJ-F10TCO-HA. The SseJ-F10TCO-HA secreted into the cytoplasm of HeLa cells was labeled with JF646-Tz (Figure 5A,B, red) and clearly co-localized with the HA-tag (green). The observation that the two labels overlapped with one another (one a fluorescent dye, the other labeled by immunostaining), further emphasizes the specificity of GCE labeling.

To ensure that the fluorescence signal observed in those HeLa cells was specific only to the secreted effector SseJ, we infected cells with wild-type Salmonella (carrying psseJ-HA) in the presence of TCO*A and pEVOL-PylRS-AF. A click dye was used to observe whether there was any background labeling in the HeLa cells. SseJ was released into the cytoplasm of the host cell, with no intracellular or non-specific fluorescent signals (Figure 5C), demonstrating that the fluorescence signal was specific to SseJ. After determining that ncAAs were incorporated specifically at an amber codon, and that secreted SseJ could be visualized in the host cell via the click reaction, the next goal was to create a super-resolution image of secreted SseJ in HeLa cells (Figure 6). In Figure 6, we demonstrate the power of GCE and the use of the JF646 dye to generate site-specific super-resolution images of the Salmonella secreted effector SseJ in HeLa cells.

Figure 1
Figure 1Scheme for site-specific fluorescent labeling of SPI-2 effectors. (A) TCO*A and JF-646-tetrazine. (B) The schematic depicts the incorporation of an ncAA in an SPI-2 effector. In the bacterial cell, a plasmid containing the gene for an effector POI (purple) and an orthogonal suppressor tRNA (red)/aminoacyl synthetase (green) pair are introduced. A native codon is swapped with an amber (TAG, red) stop codon in the effector gene sequence at a permissive location. The ncAA (dark red circle) is simply introduced into the growth medium. It is then picked up by the cell and reaches the cytosol, where it is linked to the orthogonal tRNA by the aminoacyl-tRNAsynthetase(aaRS). The ncAA-acylated tRNA with a CUA anticodon enters the ribosomal machinery as a result of the complementary amber codon on the effector mRNA (purple), incorporating the attached ncAA into the effector. The ncAA is carried by the full-length polypeptide chain of the effector site, which then folds and assembles into a functional effector protein. The newly produced effector is translocated into the host cell through the T3SS. An externally supplied fluorophore can be used to label a secreted SPI-2 effector integrated with ncAA. (C) Copper-free click reaction scheme with a fluorogenic tetrazine dye. Through the SPIEDAC click reaction, an ncAA with a strained alkene group incorporated into a POI (SseJ) reacts with a tetrazine-coupled dye (JF646-Tz). The fluorogenic, tetrazine-coupled dye JF646-Tz becomes fluorescent (red) only after successful labeling. Abbreviations: TCO*A = trans-cyclooct-2-en-L-Lysine; ncAA = non-canonical amino acid; T3SS = type three secretion system; SPI-2 = Salmonella pathogenicity island 2; POI = protein of interest; SPIEDAC = strain-promoted inverse electron-demand Diels-Alder cycloaddition. Please click here to view a larger version of this figure.

Figure 2
Figure 2:TCO*A is site-specifically incorporated into SseJ-F10TCO-HA expressed in E. coli. (A) Coomassie-stained SDS-PAGE confirms the selective incorporation of TCO*A into SseJ-F10TCO-HA (indicated by red arrow) in E. coli. (B) Expression of SseJ-F10TCO-HA in E. coli analyzed by fluorescence microscopy in the absence (top) or presence (bottom) of 1 mM TCO*A. E. coli cells expressing SseJ-F10TCO-HA in the absence or presence of TCO*A are incubated with BDP-Fl-tetrazine and imaged for BDP fluorescence (green). SseJ fluorescence (green) is only observed in the presence of the incorporated TCO*A label; note the absence of background fluorescence in the top panel. Scale bar = 2 µm. Abbreviations: TCO*A = trans-cyclooct-2-en-L-Lysine; BF = brightfield; HA = hyaluronic acid. Please click here to view a larger version of this figure.

Figure 3
Figure 3: TCO*A is site-specifically incorporated into SseJ-F10TCO-HA expressed in Salmonella. Fluorescence microscopy is used to examine the expression of SseJ-F10TCO-HA in Salmonella in the absence (top) or presence (bottom) of 1 mM TCO*A. Salmonella expressing SseJ F10TCO-HA are treated with JF646-Tz and imaged for JF646 fluorescence (magenta), in the presence or absence of TCO*A. Fluorescence of SseJ (magenta) is detected only when TCO*A is present. Scale bar = 2 µm. Abbreviations: TCO*A = trans-cyclooct-2-en-L-Lysine; BF = brightfield; HA = hyaluronic acid. Please click here to view a larger version of this figure.

Figure 4
Figure 4: Secreted SseJ-J10TCO-HA is functional. (A) HeLa cells are infected with Salmonella harboring psseJ-HA for 16 h, fixed, and immunostained with anti-HA antibody (red), LPS (green), and DAPI (blue). As expected, the formation of SseJ-dependent SIFs is observed in infected cells. (B) In the absence of TCO*A, the amber codon is not suppressed, and SseJ-dependent SIFs are absent in infected HeLa cells. HeLa cells are infected with Salmonella expressing SseJ-F10TCO-HA in the absence of TCO*A for 16 h, fixed, and immunostained with anti-HA antibody (red), LPS (green), and DAPI (blue). (C) SseJ-dependent SIFs are observed in the presence of TCO*A. Infected HeLa cells with Salmonella expressing SseJ-F10TCO-HA in the presence of 400 µM TCO*A are fixed and immunostained with anti-HA antibody SseJ (red), LPS (green), and DAPI (blue). SseJ-dependent SIFs are observed in the left panel, clearly indicating that SseJ was rescued by the expression of sseJ-F10TCO-HA using GCE. Scale bar = 10 µm. Abbreviations: HA = hyaluronic acid; LPS = lipopolysaccharide; DAPI = 4',6-diamidino-2-phenylindole; TCO*A = trans-cyclooct-2-en-L-Lysine; SIFs = Salmonella-induced filaments; GCE = genetic code expansion. Please click here to view a larger version of this figure.

Figure 5
Figure 5Labeling specificity of SseJ with JF646-Tz dye. (A) HeLa cells are infected with Salmonella expressing SseJ-F10TCO-HA in the presence of 400 µM TCO*A. TCO-tagged secreted SseJ-F10TCO-HA is labeled with 2 µM JF646-Tz in 1x PBS containing 1% casein sodium salt from bovine milk, extensively washed, fixed, and immunostained with anti-HA antibody to visualize the secreted SseJ (green). SseJ-F10TCO-HA is secreted and labeled with JF646-Tz dye (red, left panel). SseJ labeled via GCE (red) and HA (green) are clearly co-localized (right panel). (B) Using a slightly different labeling protocol, TCO-tagged secreted SseJ-F10TCO-HA is also labeled with 2 µM JF646-Tz in complete medium DMEM (without FBS), extensively washed, and fixed, and subjected to anti-HA immunofluorescence staining. Secreted SseJ-F10TCO-HA is labeled with JF646-Tz dye (red, left panel) and co-localized with HA (green). (C) To further establish that the fluorescence signal is unique to SseJ and not a product of other SPI-2 released proteins, HeLa cells are infected with wild-type Salmonella carrying psseJ-HA and pEVOL-PylRS-AF, in the presence of ncAA (TCO*A). At 16 h post infection, infected HeLa cells are treated with 2 µM JF646-Tz in complete medium (without FBS and TCO*A), and the excess dye is thoroughly removed using new growth medium. HeLa cells are PFA fixed and thoroughly washed with PBS before being immunostained for SseJ (green). The lack of visible background fluorescence signal in the left panel (JF646-Tz) indicates that almost no off-target labeling occurred within the host cells. Scale bar = 10 µm. Abbreviations: HA = hyaluronic acid; PBS = phosphate-buffered saline; FBS = fetal bovine serum; DAPI = 4',6-diamidino-2-phenylindole; TCO*A = trans-cyclooct-2-en-L-Lysine; SIFs = Salmonella-induced filaments; GCE = genetic code expansion; SPI-2 = Salmonella pathogenicity island 2; BF = brightfield. Please click here to view a larger version of this figure.

Figure 6
Figure 6Super-resolution imaging of GCE-labeled SseJ in HeLa cells. HeLa cells are infected with Salmonella expressing SseJ-F10TCO-HA in the presence of 400 µM TCO*A. TCO-tagged secreted SseJ-F10TCO-HA is labeled with 2 µM JF646-Tz under physiological conditions as described in the protocol, then extensively washed. Images are acquired using Nikon N-STORM. (A) Brightfield image of a HeLa cell under observation. Scale bar = 10 µm. (B) Diffraction-limited, widefield image of secreted SseJ labeled with TCO*A and JF-646. Scale bar = 10 µm. (C) Corresponding dSTORM image of the SseJ-decorated SIFs. Scale bar = 10 µm. C(i) Inset shows a magnified view of super-resolved SseJ in the boxed region. Scale bar = 1 µm. Abbreviations: HA = hyaluronic acid; TCO*A = trans-cyclooct-2-en-L-Lysine; SIFs = Salmonella-induced filaments; GCE = genetic code expansion; WF = widefield; BF = brightfield. Please click here to view a larger version of this figure.

Table 1. Solutions. Please click here to view a larger version of this table.

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The approach described herein was used to track the precise location of effector proteins injected into the host cell by the bacterial T3SS after infection. T3SSs are employed by intracellular pathogens such as Salmonella, Shigella, and Yersinia to transport virulence components into the host. The development of super-resolution imaging technologies has made it possible to visualize virulence factors at a previously unimaginable resolution12,13,24. However, certain labeling constraints precluded more in-depth investigation, as photoactivatable protein fusions block the T3SS pore and are not secreted. In addition, clustering artifacts and erroneous analysis can result from some fusion protein's inherent propensity to cluster or aggregate.

In some instances, the fusion protein is also cleaved, as we observed previously with PAmCherry-SsaP12,13. All these constraints are overcome by GCE, which allows for site-specific fluorescent labeling of POIs. It also allows the visualization of proteins that are not plentiful12,13. While there are a number of factors that can affect the efficiency of ncAA incorporation using an orthogonal tRNA/aminoacyl-tRNA synthetase system, it seems that the codon context is the most crucial. The orthogonal tRNA incorporates an ncAA most efficiently when the preferred codon context is AATTAGACT33. In Figure 5, we can compare the crisp labeling resulting from GCE and a site-specific fluorophore (as well as the absence of background) compared to immunostaining of an HA-tag.

As a result, researchers will be able to visualize POIs in pathogens, commensal bacteria, and eukaryotic hosts using the method outlined here. In short, genetically encoded ncAAs provide an alternate approach to effector protein labeling when conventional methods fail. However, one major limitation of this method is the inherent chemical properties of the fluorophore, such as membrane permeability, distribution, and retention, which can impact the efficiency of fluorescent labeling. Fluorescent ncAAs are an alternative option to avoid click reactions12, however these can only be visualized using two-photon microscopy. Readers are referred to a list of cell-membrane-permeable/impermeable dyes cited herein12,38,39,40,41.

In conclusion, we site-specifically labeled and visualized Salmonella secreted effector SseJ by genetically encoding ncAAs in combination with a fluorogenic fluorophore without impairing its biological function. Labeling of SseJ with JF646-Tz was highly specific, and super-resolution imaging can further be employed to visualize secreted effectors inside host cells. Thus, fluorescent labeling of bacterial effectors using dSTORM-compatible dyes and genetic code expansion allowed the subcellular spatial localization of bacterial secreted effectors in host cells, at a resolution below the diffraction limit. As this labeling platform is compatible with live-cell imaging single particle tracking, our method will make it possible to analyze effector proteins of the T3SS system with unprecedented levels of temporal and spatial resolution, which will enhance our molecular understanding of bacterial pathogenesis.

While there are certain limitations, such as poor ncAA incorporation efficiency and the photophysical constraints of the organic fluorophore, we have demonstrated that this approach can help researchers observe and localize a secreted effector inside host cells, even when it is scarce12,13. We anticipate that adaptation of the technology will open up new and exciting opportunities for imaging and research on other effector proteins produced by bacteria during infection. The method is also easily adapted for the labeling of viruses, demonstrating its broad utility.

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The authors declare that they have no competing financial interests.


This work was supported by start-up funds from the University of Texas Medical branch, Galveston, TX, and a Texas STAR award to L.J.K. We thank Prof. Edward Lemke (European Molecular Biology Laboratory, Heidelberg, Germany) for plasmid pEVOL-PylRS-AF. Images in Figure 1 were created using BioRender.


Name Company Catalog Number Comments
10x Tris/Glycine SDS running buffer BIO-RAD 1610732
Ammonium chloride Fischer Scientific A661-500
Ammonium sulphate Fischer Scientific BP212R-1
Ampicillin Sodium Sigma-Aldrich A0166 5 g
Antibiotic-Antimycotic (100x) ThermiFischer Scientific 15240096
Arabinose Sigma-Aldrich A3256 500 g
Avanti J-26XP (High-Performance Centrifuge) Beckman Coulter
Bacto-Agar BD Diagnostics, Franklin Lakes, USA 214010
BDP-FL-tetrazine Lumiprobe (USA) 2.14E+02
β-mercaptoethanol Millipore 444203 250 mL
Bromophenol blue Sigma-Aldrich B8026
BSA Sigma-Aldrich A4503 500 g
Casein Sigma-Aldrich C8654
Catalase Sigma-Aldrich C9322
Chloramphenicol Sigma-Aldrich C1919
Click Amino Acid / trans-Cyclooct-2-en – L - Lysine (TCO*A) SiChem GmbH SC-8008 Size: 500 mg
DAPI (Hoechst33342) Invitrogen H3570
DeNovix DS-11+ Spectrophotometer DeNovix
DMEM* Corning 10-013-CV * Used for maintaining HeLa Cell
DMEM** Gibco 11965-092 **Used for bacterial infection in presence of ncAA, see section 5.4.
DMSO Sigma-Aldrich D8418 250 g
Donkey anti-rabbit Alexa fluoro555 secondary antibody Invitrogen A-31572
DPBS, 1x Corning 21-031-CV
E. coli strain BL21 (DE3) Novagen (Madison, WI)
EMCCD Camera Andor iXon Ultra 897-BV
Eppendorf Safe-Lock Tube 1.5 mL (PCR clean) Eppendorf, Hamburg, Germany 30123.328
Fetal Bovine Serum (FBS) Fischer 10082147
Fisherbrand Syringe Filters - Sterile (PVDF 0.22 µm) Fischer Scientific 97203 Pack of 100
Gene Pulser Xcell Electroporator BIO-RAD 1652660
Gentamycin Sigma-Aldrich G1272 10 mL
Gibco L-Glutamine (200 mM), 100x Fischer Scientific 25-030-081
Glucose oxidase Sigma-Aldrich G7141-50KU
Glycerol Fischer Scientific BF229-4
HeLa cells ATTC CCL-2
HEPES Buffer Corning 25-060-C1 100 mL
Hydrocloric acid Fischer Scientific A144-212
ImageJ Image processing and analysis:  http://rsbweb.nih.gov/ij
IntantBlue Expedeon ISB1L Coomassie-based stain
Isopropyl β-D-1-thiogalactopyranoside (IPTG) Sigma-Aldrich I5502
Janelia Fluoro 646-tetrazine Tocris Bioscience 7279
Kanamycin Sigma-Aldrich 60615 5 g
LB Broth BD Difco 244620 500 g
Lysozyme Sigma-Aldrich L6876
μManager (v. 1.4.2) https://micro-manager.org/Download_Micro-Manager_Latest_Release
MES Sigma-Aldrich M3671 250 g
Micro pulser cuvette BIO-RAD 165-2086 0.2 cm electrode gap, pkg. of 50
Nikon N-STORM Nikon Instruments Inc. https://www.microscope.healthcare.nikon.com/products/super-resolution-microscopes/n-storm-super-resolution
Nunc EasYFlask Cell Culture Flasks ThermiFischer Scientific 156499
Omnipur Casamino Acid Calbiochem 2240 500 g
Paraformaldehhyde (PFA) Electron Microscopy Sciences  15710
PBS (10x) Roche 11666789001
Penicillin-Streptomycin Solution (100x) GenDEPOT  CA005-010 100 mL
pEVOL-PylRS-AF For plasmid construction and map see following references
1. Angew Chem Int Ed Engl. 2011, 50(17), 3878-81.
2. Angew Chem Int Ed Engl., 2012, 51, 4166-70.
Plasmid Mini-prep Kit Qiagen 27106
plasmid pWSK29-sseJ10TAG-HA Ref.: elife. 2021, 10, e67789.
plasmid pWSK29-sseJ-HA Ref.: elife. 2021, 10, e67789.                                                                   Vector map of PWSK29: https://www.addgene.org/172972/
Pluronic F-127 Millipore 540025 Protein grade, 10% Solution
Potassium phosphate monobasic Fischer Scientific P285-500
Potassium sulphate Acros Organic 424220250
Protease Inhibitor Cocktail Set I - Calbiochem Sigma-Aldrich 539131 100x Solution
Rabbit anti-HA primary antibody Sigma-Aldrich H6908
S. enterica. serovar Typhimurium 14028s Ref.: PLoS Biol. 2015, 13, e1002116.
Saponin Sigma-Aldrich 47036-50G-F
Sodium dodecyl sulfate (SDS) Sigma-Aldrich L3771
Sodium hydroxide Fischer Scientific SS255-1
Sodium Pyruvate (100 mM), 100x Corning 25-060-C1 100 mL
Sonic Dismembrator Model 100 Fischer Scientific 24932
STED microsocpe (Leica TCS SP8 STED 3X system) Leica Microsystems, Wetzlar, Germany https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-sted-one/
ThunderSTORM https://zitmen.github.io/thunderstorm/
Trizma Base Sigma-Aldrich T1503
Trypsin-EDTA (1x), 0.25%   GenDEPOT  CA014-010
Tween-20 Sigma-Aldrich P9416 100 mL
X-well tissue culture chamber slides SARSTEDT 94.6190.802



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Singh, M. K., Kenney, L. J. Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion. J. Vis. Exp. (192), e64382, doi:10.3791/64382 (2023).More

Singh, M. K., Kenney, L. J. Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion. J. Vis. Exp. (192), e64382, doi:10.3791/64382 (2023).

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