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.

1. Plasmid construction
<ol>
	<li>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.&#160;</li>
	<li>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 previou...

<ol>
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The authors declare that they have no competing financial interests.

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,...

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&#39;-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, &#945;, &#946;-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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Tris/Glycine SDS running buffer</td>
			<td>BIO-RAD</td>
			<td>1610732</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium chloride</td>
			<td>Fischer Scientific</td>
			<td>A661-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium sulphate</td>
			<td>Fischer Scientific</td>
			<td>BP212R-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin Sodium</td>
			<td>Sigma-Aldrich</td>
			<td>A0166</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>Antibiotic-Antimycotic (100x)</td>
			<td>ThermiFischer Scientific</td>
			<td>15240096</td>
			<td></td>
		</tr>
		<tr>
			<td>Arabinose</td>
			<td>Sigma-Aldrich</td>
			<td>A3256</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Avanti J-26XP (High-Performance Centrifuge)</td>
			<td>Beckman Coulter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-Agar</td>
			<td>BD Diagnostics, Franklin Lakes, USA</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>BDP-FL-tetrazine</td>
			<td>Lumiprobe (USA)</td>
			<td>2.14E+02</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-mercaptoethanol</td>
			<td>Millipore</td>
			<td>444203</td>
			<td>250 mL</td>
		</tr>
		<tr>
			<td>Bromophenol blue</td>
			<td>Sigma-Aldrich</td>
			<td>B8026</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma-Aldrich</td>
			<td>A4503</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C8654</td>
			<td></td>
		</tr>
		<tr>
			<td>Catalase</td>
			<td>Sigma-Aldrich</td>
			<td>C9322</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Sigma-Aldrich</td>
			<td>C1919</td>
			<td></td>
		</tr>
		<tr>
			<td>Click Amino Acid / trans-Cyclooct-2-en &#8211; L - Lysine (TCO*A)</td>
			<td>SiChem GmbH</td>
			<td>SC-8008</td>
			<td>Size: 500 mg</td>
		</tr>
		<tr>
			<td>DAPI (Hoechst33342)</td>
			<td>Invitrogen</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>DeNovix DS-11+ Spectrophotometer</td>
			<td>DeNovix</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM*</td>
			<td>Corning</td>
			<td>10-013-CV</td>
			<td>* Used for maintaining HeLa Cell</td>
		</tr>
		<tr>
			<td>DMEM**</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td>**Used for bacterial infection in presence of ncAA, see section 5.4.</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td>250 g</td>
		</tr>
		<tr>
			<td>Donkey anti-rabbit Alexa fluoro555 secondary antibody</td>
			<td>Invitrogen</td>
			<td>A-31572</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS, 1x</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>E. coli strain BL21 (DE3)</td>
			<td>Novagen (Madison, WI)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>EMCCD Camera</td>
			<td>Andor</td>
			<td>iXon Ultra 897-BV</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf Safe-Lock Tube 1.5 mL (PCR clean)</td>
			<td>Eppendorf, Hamburg, Germany</td>
			<td>30123.328</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum (FBS)</td>
			<td>Fischer</td>
			<td>10082147</td>
			<td></td>
		</tr>
		<tr>
			<td>Fisherbrand Syringe Filters - Sterile (PVDF 0.22 &#181;m)</td>
			<td>Fischer Scientific</td>
			<td>97203</td>
			<td>Pack of 100</td>
		</tr>
		<tr>
			<td>Gene Pulser Xcell Electroporator</td>
			<td>BIO-RAD</td>
			<td>1652660</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamycin</td>
			<td>Sigma-Aldrich</td>
			<td>G1272</td>
			<td>10 mL</td>
		</tr>
		<tr>
			<td>Gibco L-Glutamine (200 mM), 100x</td>
			<td>Fischer Scientific</td>
			<td>25-030-081</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose oxidase</td>
			<td>Sigma-Aldrich</td>
			<td>G7141-50KU</td>
			<td></td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Fischer Scientific</td>
			<td>BF229-4</td>
			<td></td>
		</tr>
		<tr>
			<td>HeLa cells</td>
			<td>ATTC</td>
			<td>CCL-2</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES Buffer</td>
			<td>Corning</td>
			<td>25-060-C1</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Hydrocloric acid</td>
			<td>Fischer Scientific</td>
			<td>A144-212</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td></td>
			<td></td>
			<td>Image processing and analysis:&#160; http://rsbweb.nih.gov/ij</td>
		</tr>
		<tr>
			<td>IntantBlue</td>
			<td>Expedeon</td>
			<td>ISB1L</td>
			<td>Coomassie-based stain</td>
		</tr>
		<tr>
			<td>Isopropyl &#946;-D-1-thiogalactopyranoside (IPTG)</td>
			<td>Sigma-Aldrich</td>
			<td>I5502</td>
			<td></td>
		</tr>
		<tr>
			<td>Janelia Fluoro 646-tetrazine</td>
			<td>Tocris Bioscience</td>
			<td>7279</td>
			<td></td>
		</tr>
		<tr>
			<td>Kanamycin</td>
			<td>Sigma-Aldrich</td>
			<td>60615</td>
			<td>5 g</td>
		</tr>
		<tr>
			<td>LB Broth</td>
			<td>BD Difco</td>
			<td>244620</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Lysozyme</td>
			<td>Sigma-Aldrich</td>
			<td>L6876</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;Manager (v. 1.4.2)</td>
			<td></td>
			<td></td>
			<td>https://micro-manager.org/Download_Micro-Manager_Latest_Release</td>
		</tr>
		<tr>
			<td>MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>250 g</td>
		</tr>
		<tr>
			<td>Micro pulser cuvette</td>
			<td>BIO-RAD</td>
			<td>165-2086</td>
			<td>0.2 cm electrode gap, pkg. of 50</td>
		</tr>
		<tr>
			<td>Nikon N-STORM</td>
			<td>Nikon Instruments Inc.</td>
			<td></td>
			<td>https://www.microscope.healthcare.nikon.com/products/super-resolution-microscopes/n-storm-super-resolution</td>
		</tr>
		<tr>
			<td>Nunc EasYFlask Cell Culture Flasks</td>
			<td>ThermiFischer Scientific</td>
			<td>156499</td>
			<td></td>
		</tr>
		<tr>
			<td>Omnipur Casamino Acid</td>
			<td>Calbiochem</td>
			<td>2240</td>
			<td>500 g</td>
		</tr>
		<tr>
			<td>Paraformaldehhyde (PFA)</td>
			<td>Electron Microscopy Sciences&#160;</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Roche</td>
			<td>11666789001</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin Solution (100x)</td>
			<td>GenDEPOT&#160;</td>
			<td>CA005-010</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>pEVOL-PylRS-AF</td>
			<td></td>
			<td></td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Plasmid Mini-prep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td></td>
		</tr>
		<tr>
			<td>plasmid pWSK29-sseJ10TAG-HA</td>
			<td></td>
			<td></td>
			<td>Ref.: elife. 2021, 10, e67789.</td>
		</tr>
		<tr>
			<td>plasmid pWSK29-sseJ-HA</td>
			<td></td>
			<td></td>
			<td>Ref.: elife. 2021, 10, e67789.&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; Vector map of PWSK29: https://www.addgene.org/172972/</td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Millipore</td>
			<td>540025</td>
			<td>Protein grade, 10% Solution</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic</td>
			<td>Fischer Scientific</td>
			<td>P285-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium sulphate</td>
			<td>Acros Organic</td>
			<td>424220250</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Set I - Calbiochem</td>
			<td>Sigma-Aldrich</td>
			<td>539131</td>
			<td>100x Solution</td>
		</tr>
		<tr>
			<td>Rabbit anti-HA primary antibody</td>
			<td>Sigma-Aldrich</td>
			<td>H6908</td>
			<td></td>
		</tr>
		<tr>
			<td>S. enterica. serovar Typhimurium 14028s</td>
			<td></td>
			<td></td>
			<td>Ref.: PLoS Biol. 2015, 13, e1002116.</td>
		</tr>
		<tr>
			<td>Saponin</td>
			<td>Sigma-Aldrich</td>
			<td>47036-50G-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium dodecyl sulfate (SDS)</td>
			<td>Sigma-Aldrich</td>
			<td>L3771</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide</td>
			<td>Fischer Scientific</td>
			<td>SS255-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100 mM), 100x</td>
			<td>Corning</td>
			<td>25-060-C1</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>Sonic Dismembrator Model 100</td>
			<td>Fischer Scientific</td>
			<td>24932</td>
			<td></td>
		</tr>
		<tr>
			<td>STED microsocpe (Leica TCS SP8 STED 3X system)</td>
			<td>Leica Microsystems, Wetzlar, Germany</td>
			<td></td>
			<td>https://www.leica-microsystems.com/products/confocal-microscopes/p/leica-tcs-sp8-sted-one/</td>
		</tr>
		<tr>
			<td>ThunderSTORM</td>
			<td></td>
			<td></td>
			<td>https://zitmen.github.io/thunderstorm/</td>
		</tr>
		<tr>
			<td>Trizma Base</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (1x), 0.25%&#160;&#160;</td>
			<td>GenDEPOT&#160;</td>
			<td>CA014-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma-Aldrich</td>
			<td>P9416</td>
			<td>100 mL</td>
		</tr>
		<tr>
			<td>X-well tissue culture chamber slides</td>
			<td>SARSTEDT</td>
			<td>94.6190.802</td>
			<td></td>
		</tr>
	</tbody>
</table>

super-resolution imaging of bacterial secreted proteins using genetic code expansion

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.

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 Figu...

Watch this Scientific Journal Video about Super-Resolution Imaging of Bacterial Secreted Proteins Using Genetic Code Expansion at JoVE.com

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

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 ...

Genetic code expansion, or GCE, overcomes limitations of other labeling strategies. It enables us to site specifically labeled proteins, and we have bright organic fluorophores that enable their visualization, so we can avoid strategies that make, say, fluorescent protein fusions, which can disrupt protein function. This method can be applied to any other system.For example, we've used it to label Zika CO protein, and that enables us to provide and produce labeled Zika virus that we can then observe infecting host cells. To begin, transfer 100 microliters of primary culture into five milliliters of LB medium containing 35 micrograms per milliliter chloramphenicol and 100 micrograms per milliliter ampicillin. Incubate at 37 degrees Celsius with shaking at 250 RPM until the optical density at 600 nanometers reaches 0.6.Replace the LB medium with the modified N-minimal medium supplemented with one millimolar TCO, a non-canonical amino acid. And grow the bacteria at 34 degrees Celsius for 30 minutes. Add 0.2%arabinose, 25 milligrams per milliliter of chloramphenicol, and 100 milligrams per milliliter of ampicillin, and grow the cells for another six hours, shaking at 250 RPM.After six hours, wash the bacteria four times over an interval of 30 minutes with fresh MgM media without non-canonical amino acids or, ncAA. Centrifuge the bacteria, re-suspend in PBS buffer, and incubate for one hour at four degrees Celsius in the dark to remove excess ncAAs. After one hour, centrifuge the bacteria at 3, 000 G for 15 minutes at four degrees Celsius, and store them for further use.For a control experiment, repeat the same experiment by expressing ncAA bearing proteins in the absence of ncAA. Re-suspend salmonella cells expressing SseJ incorporated with TCO in the absence or presence of TCO in PBS. Adjust the optical density at 600 nanometers to four in PBS, and incubate the cells with 20 micromolar Janelia Fluor 646 tetrazene or 20 micromolar BDPFL tetrazine at 37 degrees Celsius in the dark, and shake for one to two hours at 250 RPM.Pellet the cells and wash three to four times with PBS containing 5%DMSO and 0.2%Pluronic F-127. Re-suspend the pellet in PBS containing 5%DMSO. After incubating overnight at four degrees Celsius in the dark, wash twice again with PBS.Image the cells immediately using a confocal microscope, or fix them with 1.5%paraformaldehyde in PBS for 30 to 45 minutes at room temperature in the dark. Culture and maintain HeLa cells at 37 degrees Celsius, 5%carbon dioxide, and 95%humidity in high glucose DMEM supplemented with 10%FBS in addition to penicillin streptomycin. Culture bacteria one day before infection and inoculate a single colony of wild-type salmonella harboring pEVOL plasmid containing orthogonal tRNA synthetase pair and pWSK29 plasmid containing SseJ gene in five millimeters of antibiotic containing standard LB broth overnight at 37 degrees Celsius with shaking at 250 RPM.Repair a diluted cell stock at 100, 000 cells per milliliter and seed 50, 000 HeLA cells per well in 500 microliters of DMEM containing 10%FBS growth medium in an eight well chamber slide. Keep the chamber slide in an incubator for 24 hours at 37 degrees Celsius, 5%carbon dioxide, and 95%humidity. For the bacterial infection, subculture wild-type salmonella harboring pEVOL plasmid and pWSK29 plasmid containing SseJ gene by diluting 100 microliters of overnight bacterial culture into three milliliters of LB medium containing 35 micrograms per milliliter chloramphenicol and 100 micrograms per milliliter ampicillin.Incubate at 37 degrees Celsius with shaking at 250 RPM for five to seven hours. To initiate the HeLa cell infection, after taking out the HeLa cells from the incubator, wash the cells with prewarmed DPBS, and add 500 microliters of fresh DMEM containing 10%FBS to each well. Place the chamber slide back in the carbon dioxide incubator until the infection begins.After five to seven hours of incubation, dilute the salmonella culture so that the optical density at 600 nanometers is 0.2 in one milliliter of DMEM growth medium, and add the requisite amounts of the salmonella inoculum to each well of the chamber slide, so that the multiplicity of infection is 100. After incubating the infected cells in a carbon dioxide incubator for 30 minutes, wash the cells three times with prewarmed DPBS to remove extracellular salmonella. Set this time point to zero hours post-infection, and add 500 microliters of complete medium containing 100 micrograms per milliliter of gentamicin for one hour.After incubation, again, wash the cells three times with DPBS as shown earlier and add 500 microliters of the complete medium supplemented with 0.2%arabinose, 10 micrograms per milliliter of gentamicin to each well of the chamber slide. In a control experiment, perform similar infections of HeLa cells without TCO. After incubating the chamber slide for 10 hours in the carbon dioxide incubator, replace the complete medium with fresh complete medium without TCO, and wash the HeLa cells four times over an interval of 30 minutes each with prewarmed DPBS.Then wash the cells with fresh complete medium without TCO. After 12 hours post-infection, aspirate off the medium from the cells and wash the cell with prewarmed DPBS twice or three times. In one group of the wells, add 500 microliters of the dye solution mixture one, and in another group of the wells of the same chamber slide, add 500 microliters of the dye solution mixture two.Place the chamber slides back into the carbon dioxide incubator for one and 1/2 to two hours. After 13.5 to 14 hours post-infection, rinse the HeLa cells twice with prewarmed DPBS, and add 500 microliters of fresh DMEM supplemented with FBS. After incubating for 30 minutes, wash the cells again with prewarmed DPBS as shown earlier, and then wash with fresh DMEM four times over an interval of 30 minutes.At 16 hours post-infection, fix the HeLa cells with paraformaldehyde by adding 200 microliters of 4%paraformaldehyde to each well and incubating for 10 minutes at room temperature in the dark. Aspirate off the paraformaldehyde. Rinse three times with PBS, and store the cells in PBS at four degrees Celsius in the dark.Bring the cells to the microscope, and place the imaging chamber on the microscope stage in the sample holder. Then change the medium in the well with 0.4 milliliters of the freshly made GLOX BME imaging buffer. Decrease the laser power to around one milliwatt to identify a HeLa cell of interest and adjust the focal plane and laser beam angle while illuminating the sample with low 647 nanometer laser densities.Adjust the HILO illumination angle. Capture a reference to fraction limited image of the target structure, and switch the fluorophores to the dark state by turning the laser to its maximum power. Set the pre-amplifier gain to three and activate the frame transfer.Then set EM gain to 200 for higher sensitivity in dSTORM measurements. Adjust the laser strength to a suitable level where blinking events are separated in space and time. Set the exposure time to 30 milliseconds and begin the acquisition.Acquire 10, 000 to 30, 000 frames. Repeat the similar acquisition at different ROIs. For the imagery reconstruction of dSTORM, open image J and import the raw data.Open the Thunderstorm plugin, and configure the camera setting corresponding to the device. Go to run analysis and set the appropriate settings, such as image filtering, localization methods, and subpixel localization of molecules. Click Okay to start the image reconstruction and begin the post-processing end result analysis.Salmonella expressing SseJ incorporated with TCO are treated with Janelia Fluor 646 tetrazine and imaged with Janelia Fluor 646 fluorescence in the absence or presence of TCO. The fluorescence of SseJ is detected only when TCO is present. HeLa cells are infected with salmonella harboring psseJ-HA for 16 hours, fixed and immunostained with anti HA antibody, LPS, and DAPI.As expected, SseJ dependent salmonella induced filaments, or SIFs, are formed in the infected cells. In the absence of TCO, the amber codon is not suppressed, and SseJ dependent SIFs are absent in infected HeLa cells. SseJ dependent SIFs are observed in the presence of TCO, which clearly indicates that SseJ was rescued by the expression of SseJF10TCOHA using genetic code expansion, or GCE.Labeling specificity of secreted SseJF10TCOHA in HeLa cells through SPIEDAC click reactions using two alternate dye mixture one and dye mixture two are shown here. Click dye Janelia Fluor 646 TZ was further used to observe whether there was any background labeling in HeLa cells infected with wild-type Salmanella carrying SseJ HA in the presence of TCO and P vault plasmid. SseJ was released into the cytoplasm of the host cell with no intracellular or non-specific fluorescent signals demonstrating that the fluorescent signal was specific to SseJ.Super resolution imaging of GCE labeled SseJ in HeLa cells is presented in this figure. A brightfield image of a HeLa cell under observation is shown here. The diffraction limited widefield image of secreted SseJ labeled with TCO and Janelia Fluor 646 and the corresponding dSTORM image of the SseJ decorated SIFs are presented here.The insert shows a magnified view of a super resolved SseJ in the boxed region. NCAA stock solutions were prepared in NaOH. Do not forget to neutralize it before or after addition to the media.After labeling protein of interest, wash the cell extensively, so that background signal is minimal. Following our GCE procedure, now, we can label any virulence factor in bacteria and follow its activity using our imaging techniques.

super-resolution-imaging-bacterial-secreted-proteins-using-genetic

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JoVE Journal

Biology

1.3K Views.  University of Texas Medical Branch. L'espansione del codice genetico, o GCE, supera i limiti di altre strategie di etichettatura. Ci consente di localizzare proteine specificamente etichettate e abbiamo fluorofori organici luminosi che consentono la loro visualizzazione, in modo da evitare strategie che producono, ad esempio, fusioni proteiche fluorescenti, che possono interrompere la funzione proteica. Questo metodo può essere applicato a qualsiasi altro sistema.Ad esempio, lo abbiamo usato per etichettare la proteina ...