This work was in part supported by grants from the Bill and Melinda Gates Foundation (OPP1109789), the National Natural Science Foundation of China (31570129), and start-up funds from the Tsinghua University School of Medicine to B.J. B.J. is a Wellcome Trust Investigator (207487/Z/17/Z).

1. Renilla-Firefly Dual-luciferase Reporter
NOTE: For a visual representation of this method, see Figure 2.
<ol>
	<li>Inoculate mycobacterial reporter strains from -80 &#176;C stock with 2 mL of 7H9 medium. Wild-type dual-luciferase, as well as mutated Renilla (reporter) strains, needs to be used to allow the calculation of mistranslation rates (see step 1.7). Shake at 37 &#176;C for 1 to 2 days till OD600&#160;reaches the stationary phase (OD &#62; 3).</li>
	<li>Aliquot and dilute to OD600nm around 0.1 - 0.5. For typical experiments, use three independent biological replicate cultures. Anhydrotetracycline (ATC), a tetracycline analog inducer of reporter expression (which is controlled by a tetracycline-inducible promoter) to a final concentration of 50 ng/mL.</li>
	<li>To measure effects of kasugamycin on mistranslation rates21, add different doses of kasugamycin to the culture (see Figure 4 for the indicated doses) at the same time (at the tested doses, kasugamycin has no antimicrobial activity). Note that it is important to include at least one non-induced control for each reporter. Culture-induce cultures for 4-6 h at 37 &#176;C with shaking.</li>
	<li>Transfer the bacterial cultures to a 2 mL tube, and centrifuge at 3,220 x g for 5 min at room temperature to pellet down the bacteria. Discard the supernatant.</li>
	<li>Disrupt the bacteria by adding 40 &#956;L of 1x passive lysis buffer (supplied by a dual-luciferase kit), which has been diluted (1:1) in double-distilled water. Transfer the resuspended bacterial lysate to a white 96-well plate, one well per sample, and shake at room temperature for 20 min. 
	CAUTION: Do not over incubate the bacteria in lysis buffer (for more than 30 min).</li>
	<li>Add 80 &#956;L of firefly substrate to each well, shake for 15 s, and measure the luminescence by luminometer with 1,000 ms as integration time. Use either an automated injector or a multichannel pipette to avoid pipetting errors.</li>
	<li>Add 80 &#956;L of Renilla substrate to each well, shake for 15 s, and measure the luminescence by luminometer with 1,000 ms as integration time.</li>
	<li>Subtract the background luminescence - measured using either wild-type M. smegmatis (i.e., not containing reporters) or uninduced reporter lysate - from the measured values. Use the corrected values to calculate the mistranslation rates of each condition by using the following equation11,25, where DN refers to the activity in the mutated reporter strain: 
	<img alt="Equation 1" src="/files/ftp_upload/59453/59453eq1.jpg" /></li>
</ol>
2. Nluc/GFP Reporter
NOTE: For a visual representation of this method, see Figure 3.
<ol>
	<li>Inoculate the bacterial reporter strain from -80 &#176;C stock with 2 mL of 7H9 medium. Shake at 37 &#176;C for 1 to 2 days till OD600&#160;reaches the stationary phase (OD &#62;3).</li>
	<li>Subculture to 50 mL of 7H9 medium and grow till OD600&#160;reaches the late stationary phase (&#62;4).</li>
	<li>Before aliquoting the bacteria to a 96-well plate, add ATC at a final concentration of 50 ng/mL and mix well. Induction of the bulk culture ensures that all wells contain the same amount of inducer and that the induction of the reporter is synchronized. Aliquot the bacteria to a clear, round-bottomed 96-well plate, with 100 &#956;L of volume in each well.</li>
	<li>To screen for small molecules affecting mistranslation rates, add the compound at the indicated concentration to select wells. For the purposes of this protocol, use kasugamycin (at doses indicated in Figure 5) as an illustration. Add different doses of kasugamycin to select wells (each experimental group should contain at least two biological replicates).</li>
	<li>Shake and induce the samples at 37 &#176;C for 16-20 h. 
	NOTE: It is necessary to seal the plate with film. Also, all the edge wells need to be filled with at least 200 &#956;L of sterile water to limit evaporation from the test wells.</li>
	<li>Take 80 &#956;L from each well, using a multichannel pipette, and transfer the samples to a black 96-well plate (which maximizes the fluorescence signal measurement). Measure the GFP signal by luminometer with 20 ms as integration time.</li>
	<li>After measuring the GFP signal, centrifuge the plate at 3,220 x g for 10 min. Transfer 50 &#956;L of the supernatant to a white-bottomed 96-well plate (which maximizes the luminescence signal measurement), add 50 &#956;L of Nluc substrate to each well, mix them well, and measure the luminescence by luminometer with 1,000 ms as integration time.</li>
	<li>Determine the Nluc/GFP ratio by dividing the corrected Nluc luminescence values by GFP fluorescence: this measure (in arbitrary units) is a relative measure of aspartate for asparagine mistranslation.</li>
</ol>

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The authors have nothing to disclose.

The protocol described here can be adapted for the measurement of mistranslation rates in a wide variety of organisms. There are a number of considerations that should be kept in mind when adapting the protocol to other systems. First, the purpose of the measurement should be considered. For the accurate measurement of mistranslation rates using gain-of-function reporters, what is needed is (a) a robust readout assay (e.g., enzymatic function [in this case, luciferase activity] with a broad linear range). Also, look for (b) a loss of function mutation in a critical residue of the reporter protein that results in a very significant loss of function, below the expected rate of mistranslation. For example, if the expected mistranslation rate to be measured is approximately 10-3/codon, the loss of function mutation needs to be greater than 1,000-fold; otherwise, the lower range of mistranslation events will not be sensitively detected. Lastly, for the accurate measurement of mistranslation rates using gain-of-function reporters, (c) a robust benchmark reporter is needed &#8212; in this case, firefly luciferase for section 1 of the protocol. Ideally, the benchmark reporter should be fused to the mutated enzymatic reporter, guaranteeing (for all intents and purposes) that they are both expressed in equimolar ratios. The benchmark (and primary reporter) readout should be robust to perturbations to exposed environments. For example, the fluorescence of wild-type GFP is highly susceptible to perturbation by changes in pH28, making it unsuitable as a benchmark for the measurement of mistranslation rates in phagocytosed mycobacteria in macrophages, which reside in phagosomes that are below pH 729. On the other hand, for applications such as small molecule screening, the most important considerations for a primary screen will be the reproducibility of the measurements (i.e., precision, as opposed to accuracy), the minimization of manual handling, and small culture volumes. The accurate measurements of mistranslation rates are less important and can be performed as secondary assays. Finally, it should be noted that the reporters described in this protocol, based on the enzymatic function of luciferase enzymes, will only measure mean mistranslation rates of a bacterial population but cannot give information about single-cell heterogeneity variation in mistranslation rates. Given the importance of single-cell variability in adaptive phenotypes30,31,32, fluorescent reporters for the measurement of mistranslation events have been developed12,33, including for the measurement of mistranslation in mycobacteria5.
Having considered the requirements for the measurement of mistranslation, it should be noted that the genetic gain-of-function reporters such as described in this protocol can only measure one type of mistranslation (i.e., one amino acid substitution for one codon) per reporter. Therefore, for the measurement of different mistranslation events, multiple reporters are required. For example, for the measurement of mistranslation by ribosomal decoding errors of near-cognate codons by lysyl-tRNA at one position, at least 16 reporters are required2,3,11. High-precision mass spectrometry and bioinformatics allow for the potential identification of many different types of mistranslation events simultaneously18; however, these methods also come with caveats. In general, they are less accurate than gain-of-function reporters. Furthermore, as stated earlier, they are less suitable for the detection of certain types of mistranslation, namely those that could be conflated with nonenzymatic deamidation23. Finally, mass spectrometry is unsuitable as a read-out for medium- or high-throughput screening.
For the adaptation of these reporters and protocols for the measurement of mistranslation in other systems, further considerations include a robust expression of the reporter in the desired model system. We initially attempted to use the dual-luciferase system developed by Farabaugh and colleagues in our mycobacterial system without modifications but had no success, due to a lack of reporter expression. Extensive troubleshooting identified that both codon-optimization and a minor sequence modification of the reporters were required to allow for robust expression25 (and see above). It is conceivable that similar adaptations of the reporters would be required for use in other systems.
Finally, consideration should be given to the type of mistranslation event being measured. For example, if there is a need to measure stop-codon readthrough (nonsense suppression), the stop codon&#8217;s loss of function mutation needs to be introduced within a noncritical region of the primary reporter protein34. Otherwise, only specific nonsense suppression events that regain the critical residue (and, hence, reporter function) will be measured by the assay, which would potentially lead to a substantial underestimation of the true mistranslation rates. There is increasing evidence that translational error plays a possible adaptive role in a large number of organisms1,12,13,35. However, the measurement of mistranslation events is still limited to a small, albeit growing number of model species. The adaptation of sensitive methods to measure mistranslation has the potential to further increase scientists&#8217; understanding of the role of translation error in physiology and pathology.

The flow of information in molecular biology requires the translation of genetic information to functional proteins. As with all biological systems, gene translation also involves measurable errors. Estimates of the rates of error in translation are typically quoted as approximately 1/10,000 per codon (reviewed by Ribas de Pouplana et al.1). However, error rates vary widely, from fewer than 10-5 to more than 0.05/codon1,2,3,4,5. The wide range of error rates, spanning more than three orders of magnitude, is due to the fact that errors can arise from multiple steps in the translation pathway: from stochastic, mutational, or stress-induced errors in aminoacylation6,7,8,9,10, physiological misacylation of asparaginyl- and glutaminyl-tRNAs5, or ribosomal decoding errors2,3,11. Measurably high error rates, representing over 0.01/codon, suggested that translational errors may perform physiological functions1,12 and that mistranslation may be context-specific13.
We and others have shown that naturally occurring errors in gene translation may be adaptive, especially during environmental stress1,5,12,14,15,16,17,18. In mycobacteria, errors generated by the two-step indirect glutamine/asparagine tRNA aminoacylation pathway19,20 result in a remarkably increased tolerance for the first-line antituberculosis antibiotic rifampicin5. Therefore, we speculated that decreasing mycobacterial mistranslation with a small molecule may potentiate killing by rifampicin. We screened for and identified the naturally occurring aminoglycoside kasugamycin as a compound that could decrease mycobacterial mistranslation, potentiate rifampicin-mediated killing of mycobacteria both in vitro and in vivo21, and limit the emergence of rifampicin resistance21, which threatens the global control of tuberculosis22 &#8212; the world&#39;s most deadly pathogen.
To study translational error, methods for the measurement of mistranslation have to be employed. There are multiple methods that have been developed for the measurement of mistranslation, each with advantages and disadvantages. Briefly, precision mass spectrometry-based methods have several advantages, the most important of which is that with newer algorithms for the detection of multiple types of translational error, a relatively unbiased measurement of mistranslation can be performed18. However, mass spectrometry is not very suitable for the measurement of deamidation mistranslation events &#8212; precisely the type of mistranslation that occurs in mycobacteria due to the error-prone indirect tRNA aminoacylation pathway. This is because of high-frequency nonenzymatic deamidation that occurs in the processing of samples for mass spectrometry23, resulting in an extremely high background signal. Therefore, for the detection of errors in this pathway, genetic gain-of-function reporters offer distinct advantages. Specifically, suitable gain-of-function reporters can have extremely low background rates, allowing the measurement of very low error rates11.
Since performing experiments with pathogenic Mycobacterium tuberculosis requires specialized facilities and extra precautions, we perform most experiments in the nonpathogenic mycobacterium M. smegmatis &#8212;&#160;and have shown earlier that results between the two species are broadly comparable5,21. To measure mistranslation rates in mycobacteria generated by the indirect tRNA aminoacylation pathway, we modified a Renilla-Firefly dual-luciferase system that had been previously developed to measure ribosomal decoding errors in E. coli11 for use in mycobacteria. We made three specific modifications: the original reporter did not express efficiently in mycobacteria, and therefore, the sequence was codon-optimized, and the C-terminal three amino acids of the firefly luciferase, serine-lysine-leucine, which has been annotated as a trafficking signal in some systems24, was modified to isoleucine-alanine-valine25. The original reporter had a critical lysine residue in the firefly luciferase mutated. Instead, we mutated either a critically conserved aspartate (D120) or glutamate (E144) residue in the Renilla luciferase to asparagine and glutamine, respectively25 (Figure 1). The reporter was subcloned into the episomal tetracycline-inducible plasmid pUV-tetOR (see the Table of Materials). Gain-of-function reporters mutate critically conserved functional residues in enzymes/fluorescent proteins that renders them nonfunctional11,26. Translational (or theoretically, transcriptional) errors that synthesize the functional variant of the protein would result in measurable enzyme activity in a subset of translated proteins. To correct for variation in protein abundance, the mutated reporter is coexpressed with a functional protein that acts as a benchmark and allows for accurate quantification of gain-of-function11. Whilst the Renilla-Firefly dual-luciferase reporter allowed for the accurate measurement of specific mycobacterial mistranslation rates5,25 (Figure 2 and section 1 of the protocol), we quickly realized that it is not suitable for medium/high-throughput screening of molecules that would alter the mistranslation rate. This is due mainly to two reasons, namely, a) the relative lack of potency of Renilla luciferase meant that a minimum of 1 mL of mycobacterial culture/sample was required to measure mistranslation rates, and b) the requirement for lysis of the cells prior to enzyme activity measurement required excessive manual handling: mycobacterial cells have a thick and multi-layered cell wall and envelope that is relatively resistant to lysis. Therefore, we looked to develop a new gain-of-function reporter system that could be used with small volumes (e.g., in a 96-well plate system) and did not require cell-lysis for measurements. We used the highly potent Nluc luciferase and identified a critical aspartate residue that, when mutated to asparagine, resulted in 2 logs loss of function (Figure 1). Furthermore, the small size of Nluc allowed it to have an N-terminal secretion signal tag &#8212; from antigen 85A, a major secreted antigen in mycobacteria27 &#8212; that would allow Nluc to be secreted into culture supernatant and circumvent the requirement for cell lysis. The benchmark protein, GFP, was expressed from the same promoter as the mutated Nluc, but from an integrated vector (see the Table of Materials), and could be measured in intact cells21 (Figure 3). Despite these advantages, the Nluc/GFP reporter (section 2 of the protocol) also has disadvantages: the relatively modest reduction in Nluc activity (100-fold) with the D140N mutation would not permit the measurement of extremely low mistranslation rates, making the reporter more suitable as a screening tool than for the accurate measurements of translational error. Furthermore, Nluc has no critical glutamate residues; therefore, only asparagine-to-aspartate error rates could be measured. The general principles described in this work should allow researchers to either use these reporters as we have done or modify reporters as appropriate for an accurate and/or facile measurement of other specific translational error rates in their model system of choice.

<table border="0" cellpadding="0" cellspacing="0" style="width:655px;" width="655">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Middlebrook 7H9</td>
			<td style="width:103px;">BD Difco</td>
			<td style="width:136px;">271310</td>
			<td style="width:169px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">anhydrotetracycline</td>
			<td style="width:103px;">Cayman Chemical&#160;</td>
			<td style="width:136px;">10009542</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">kasugamycin</td>
			<td style="width:103px;">sigma</td>
			<td style="width:136px;">K4013</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Dual-luciferase reporter assay system</td>
			<td style="width:103px;">promega</td>
			<td style="width:136px;">E1960</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">Nano-Glo luciferase assay</td>
			<td style="width:103px;">promega</td>
			<td style="width:136px;">N1120</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Fluoroskan Ascent FL luminometer</td>
			<td style="width:103px;">Thermo</td>
			<td style="width:136px;">/</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Assay Plate, 96 Well White, Flat Bottom High Binding, No Lid</td>
			<td style="width:103px;">Costar</td>
			<td align="right" style="width:136px;">3922</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Assay Plate, 96 Well Black, Flat Bottom High Biding, No Lid</td>
			<td style="width:103px;">Costar</td>
			<td align="right" style="width:136px;">3925</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">96 Well Cell Culture Cluster, Flat Bottom with Lid</td>
			<td style="width:103px;">Costar</td>
			<td align="right" style="width:136px;">3599</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;"></td>
			<td style="width:103px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">Non-commercial reagents (plasmids)</td>
			<td style="width:103px;"></td>
			<td style="width:136px;"></td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:247px;">pUV-TetOR-RenFF</td>
			<td style="width:103px;">NA</td>
			<td style="width:136px;">NA</td>
			<td style="width:169px;">episomal shuttle plasmid that allows tetracycline-inducible expression of the wild-type dual luciferase reporter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">pUV-TetOR-Ren-D120N-FF</td>
			<td style="width:103px;"></td>
			<td style="width:136px;"></td>
			<td>dual-luciferase reporter with mutated Renilla</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:247px;">pUV-TetOR-Ag85ASec-Nluc-D140N</td>
			<td style="width:103px;">NA</td>
			<td style="width:136px;">NA</td>
			<td style="width:169px;">episomal shuttle plasmid with a tetracyclin-inducible secretable version of mutated Nluc luciferase</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:247px;">pMC1S-GFP</td>
			<td style="width:103px;">NA</td>
			<td style="width:136px;">NA</td>
			<td style="width:169px;">Mycobacterial integrated (L5 site) vector for tetracycline-inducible expression of GFP</td>
		</tr>
	</tbody>
</table>

measurement of specific mycobacterial mistranslation rates with gain-of-function reporter systems

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 per codon, the actual error rates vary widely, depending on the species, environment, and codons being studied. We have previously shown that mycobacteria use a two-step pathway for the generation of aminoacylated glutamine and asparagine tRNAs and that this is specifically associated with relatively high error rates due to the modulation of mistranslation rates by an essential component of the pathway, the amidotransferase GatCAB. We modified a previously employed Renilla-Firefly dual-luciferase system that had been used to measure mistranslation rates in Escherichia coli for use in mycobacteria to measure specific mistranslation rates of glutamate at glutamine codons and aspartate for asparagine codons. Although this reporter system was suitable for the accurate estimation of specific error rates, lack of sensitivity and requirements for excessive manipulation steps made it unsuitable for high-throughput applications. Therefore, we developed a second gain-of-function reporter system, using Nluc luciferase and green fluorescent protein (GFP), which is more amenable to medium/high-throughput settings. We used this system to identify kasugamycin as a small molecule that can decrease mycobacterial mistranslation. Although the reporters that we describe here have been used to measure specific types of mycobacterial mistranslation, they may be modified to measure other types of mistranslation in a number of model systems.

A cartoon illustrating the general outline of the two reporter systems used in this work are shown in Figure 1. An overview of section 1 of the protocol is shown in Figure 2 and an overview of section 2 in Figure 3. The effect of kasugamycin on mycobacterial mistranslation is shown in Figure 4, as measured by the Renilla-Firefly reporter system. To show the specificity of the kasugamycin action, the reporter was also expressed in a strain of M. smegmatis in which ksgA was deleted (&#8710;ksgA). KsgA is an rRNA dimethyl transferase, and ksgA&#173;-deleted strains are relatively resistant to kasugamycin. Kasugamycin action on mistranslation was also measured using the Nluc/GFP reporter, as shown in Figure 5.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/59453/59453fig1.jpg" /> 
Figure 1: Cartoon illustrating the two gain-of-function reporter systems. (A) The Renilla-Firefly reporter system is comprised of two luciferase enzymes expressed as a fusion protein and under the control of a tetracycline-inducible promoter. Expression of the wild-type dual enzyme results in high measurable activity of both Renilla and firefly luciferases (top). Mutation of a critical aspartate residue, D120, in Renilla to asparagine renders the Renilla enzyme (D120N) inactive, but firefly luciferase is still active. Mistranslation of asparagine to aspartate (in this case, from physiologically misacylated Asp-tRNAAsn tRNA5,21) results in a small proportion of the translated Renilla proteins gaining activity, which can be measured. Comparison of the Renilla/Firefly activity of the mutated reporter compared with the wild-type dual enzyme allows the calculation of the asparagine to an aspartate mistranslation rate. (B) The Nluc/GFP reporter system operates on a similar principle. The mutated Nluc (D140N) gene has an N-terminal secretion signal derived from antigen 85A, a major secreted mycobacterial protein. Both the nluc and gfp genes are expressed from identical tetracycline-inducible promoters, to allow the use of GFP as a relative expression benchmark. Note the lack of wild-type Nluc control strain: this reporter is primarily used in screening, where measurements of relative mistranslation rates are sufficient for the primary screen. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59453/59453fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/59453/59453fig2.jpg" /> 
Figure 2: Outline of measuring the mistranslation rate using Renilla-Firefly reporter (section 1 of the protocol). Fresh cultures of M. smegmatis, transformed with a plasmid expressing the dual-luciferase reporters, are grown. The reporter expression is induced, and investigational compounds or conditions are tested. Following 4-6 h of reporter expression, the cultures are pelleted and lysed and the lysates are tested for dual-luciferase activity. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59453/59453fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/59453/59453fig3.jpg" /> 
Figure 3: Outline of measuring the mistranslation rate Nluc-GFP reporter (section 2 of the protocol). A fresh culture of M. smegmatis, transformed with the Nluc and GFP reporters, is grown to a sufficient volume for all the plates to be tested (assuming ~10 mL/96-well plate). Immediately prior to pipetting the bacterial culture into each well, induce the expression of the reporters with ATC added to the bulk culture. Incubate the wells by shaking them overnight. To measure the relative mistranslation rates, transfer the cultures to a black plate (to increase the sensitivity of fluorescence detection), measure the GFP fluorescence, and then, spin the plates down to pellet the bacteria. Transfer the supernatant to a white plate for Nluc activity measurement. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59453/59453fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/59453/59453fig4.jpg" /> 
Figure 4: Representative result of using Renilla-Firefly reporter measuring the mistranslation rate in the presence of kasugamycin in different strains. Mistranslation rates of aspartate for asparagine in (A) wild-type M. smegmatis and (B) a strain deleted for ksgA (&#8710;ksgA) following treatment with kasugamycin (ksg) as measured by the Renilla-Firefly dual reporter. The cultures were treated with kasugamycin at indicated doses (in micrograms/milliliter) for 6 h prior to cell lysis and measurements. The corrected Ren/FF ratios (y-axes) are indicative of the aspartate for asparagine mistranslation rates. The deletion of ksgA results in a higher baseline mistranslation rate, which is more resistant to modulation by kasugamycin. The bars indicate the mean values, with standard deviation as error. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59453/59453fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/59453/59453fig5.jpg" /> 
Figure 5: Representative result of using Nluc-GFP reporter measuring the mistranslation rate in the presence of kasugamycin. Relative mistranslation rates of aspartate for asparagine in wild-type M. smegmatis as measured by the Nluc/GFP reporter, following treatment with kasugamycin (ksg) overnight (16 h). The bars indicate the mean values, with standard deviation as error. <a href="https://jove.unicartagenaproxy.elogim.com/files/ftp_upload/59453/59453fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems at JoVE.com

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

The translation of genes into proteins is prone to errors. Although the average rate of translational error in model systems is estimated to be 1/10,000 ...

measurement-specific-mycobacterial-mistranslation-rates-with-gain

Universidad de Cartagena UDEC

Research

JoVE Journal

Immunology and Infection

5.8K Views.  Tsinghua University School of Medicine. In this article, we present two complementary methods to measure specific rates of translational error and mistranslation in the model mycobacterium, Mycobacterium smegmatis, using gain-of-function reporter systems. The methods can be employed to measure accurate error rates in low throughput or relative error rates in a more high-throughput setting.

Video: Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

Estimating Virus Production Rates in Aquatic Systems

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative estimates of this process is critical as we can then develop better models of microbial community structure and function as well as advance our understanding of how viruses work to alter aquatic biogeochemical cycles. The virus reduction technique allows researchers to estimate the rate at which virus particles are released from the endemic microbial community. In brief, the abundance of free (extracellular) viruses is reduced in a sample while the microbial community is maintained at near ambient concentration. The microbial community is then incubated in the absence of free viruses and the rate at which viruses reoccur in the sample (through the lysis of already infected members of the community) can be quantified by epifluorescence microscopy or, in the case of specific viruses, quantitative PCR. These rates can then be used to estimate the rate of microbial mortality due to virus-mediated cell lysis.

The turnover rate of viruses in marine and freshwater systems can be estimated by a reduction and reoccurrence technique. The data allow researchers to infer rates of virus-mediated microbial mortality in aquatic systems.

Viruses are pervasive components of marine and freshwater systems, and are known to be significant agents of microbial mortality. Developing quantitative ...

Measurement of &#947;HV68 Infection in Mice

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human &gamma;-HVs, such as EBV and KSHV, has severely hindered detailed pathogenic studies. Murine &gamma;-herpesvirus 68 (&gamma;HV68) shares extensive genetic and biological similarities with human &gamma;-HVs and is a natural pathogen of murid rodents2. As such, evaluation of &gamma;HV68 infection of mice inbred strains at different stages of viral infection provides an important model for understanding viral lifecycle and pathogenesis during &gamma;-HVs infection.
Upon intranasal inoculation, &gamma;HV68 infection results in acute viremia in the lung that is later resolved into a latent infection of splenocytes and other cells, which may be reactivated throughout the life of the host3,4. In this protocol, we will describe how to use the plaque assay to assess infectious virus titer in the lung homogenates on Vero cell monolayers at the early stage (5 - 7 days) of post-intranasal infection (dpi). While acute infection is largely cleared 2 - 3 weeks postinfection, a latent infection of &gamma;HV68 is established around 14 dpi and maintained later on in the spleen of the mice. Latent infection usually affects a very small population of cells in the infected tissues, whereby the virus stays dormant and shuts off most of its gene expression. Latently-infected splenocytes spontaneously reactivate virus upon explanting into tissue culture, which can be recapitulated by an infectious center (IC) assay to determine the viral latent load. To further estimate the amount of viral genome copies in the acutely and/or latently infected tissues, quantitative real-time PCR (qPCR) is used for its maximal sensitivity and accuracy. The combined analyses of the results of qPCR and plaque assay, and/or IC assay will reveal the spatiotemporal profiles of viral replication and infectivity in vivo.

&#947;-Herpesviruses (&#947;-HVs) establish life-long persistency in their host. Infection of mice with &#947;-HV68 provides a genetically tractable in vivo model for the characterization of the lifecycle/pathogenesis of &#947;HVs. This protocol describes the detection and quantitation of &#947;HV68 infection at acute and latent stages following infection by plaque-forming, infectious center, and qPCR assays.

&gamma;-Herpesviruses (&gamma;-HVs) are notable for their ability to establish latent infections of lymphoid cells1. The narrow host range of human ...

Efficient Recombinant Parvovirus Production with the Help of Adenovirus-derived Systems

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 which regulate the expression of non-structural (NS1 and NS2) and capsid proteins (VP1 and VP2) respectively1. They attract high interest as anticancer agents for their oncolytic and oncosuppressive abilities while being non-pathogenic for humans2. NS1 is the major effector of viral cytotoxicity3. In order to further enhance their natural antineoplastic activities, derivatives from these vectors have been generated by replacing the gene encoding for the capsid proteins with a therapeutic transgene (e.g. a cytotoxic polypeptide, cytokine, chemokine, tumour suppressor gene etc.)4. The recombinant parvoviruses (recPVs) vector retains the NS1/2 coding sequences and the PV genome telomeres which are necessary for viral DNA amplification and packaging. Production of recPVs occurs only in the producer cells (generally HEK293T), by co-transfecting the cells with a second vector (pCMV-VP) expressing the gene encoding for the VP proteins (Fig. 1)4. The recPV vectors generated in this way are replication defective. Although recPVs proved to possess enhanced oncotoxic activities with respect to the parental viruses from which they have been generated, their production remains a major challenge and strongly hampers the use of these agents in anti-cancer clinical applications.
We found that introduction of an Ad-5 derived vector containing the E2a, E4(orf6) and the VA RNA genes (e.g. pXX6 plasmid) into HEK293T improved the production of recPVs by more than 10 fold in comparison to other protocols in use. Based on this finding, we have constructed a novel Ad-VP-helper that contains the genomic adenoviral elements necessary to enhance recPVs production as well as the parvovirus VP gene unit5. The use of Ad-VP-helper, allows production of rec-PVs using a protocol that relies entirely on viral infection steps (as opposed to plasmid transfection), making possible the use of cell lines that are difficult to transfect (e.g. NB324K) (Fig. 2). We present a method that greatly improves the amount of recombinant virus produced, reducing both the production time and costs, without affecting the quality of the final product5. In addition, large scale production of recPV (in suspension cells and bioreactors) is now conceivable.

Here we describe a protocol based only on cell infection, which improves the efficiency of recombinant parvovirus production by more than 100 fold in comparison to other protocols in use. This protocol relies on the use of a novel adenovirus 5-based helper containing the parvovirus VP transcription unit (Ad-VP).

Rodent parvoviruses (PV) such as rat H-1PV and MVM, are small icosahedral, single stranded, DNA viruses. Their genome includes two promoters P4 and P38 ...

Study of Phagolysosome Biogenesis in Live Macrophages

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome maturation is the complex and tightly regulated process during which a nascent phagosome undergoes drastic transformation through well-orchestrated interactions with various cellular organelles and compartments in the cytoplasm. This process, which is essential for the physiological function of phagocytic cells by endowing phagosomes with their lytic and bactericidal properties, culminates in fusion of phagosomes with lysosomes and biogenesis of phagolysosomes which is considered to be the last and critical stage of maturation for phagosomes. In this report, we describe a live cell imaging based method for qualitative and quantitative analysis of the dynamic process of lysosome to phagosome content delivery, which is a hallmark of phagolysosome biogenesis. This approach uses IgG-coated microbeads as a model for phagocytosis and fluorophore-conjugated dextran molecules as a luminal lysosomal cargo probe, in order to follow the dynamic delivery of lysosmal content to the phagosomes in real time in live macrophages using time-lapse imaging and confocal laser scanning microscopy. Here we describe in detail the background, the preparation steps and the step-by-step experimental setup to enable easy and precise deployment of this method in other labs. Our described method is simple, robust, and most importantly, can be easily adapted to study phagosomal interactions and maturation in different systems and under various experimental settings such as use of various phagocytic cells types, loss-of-function experiments, different probes, and phagocytic particles.

Phagocytic cells play a major role in the innate immune system by removing and eliminating invading microorganisms in their phagosomes. Phagosome ...

Visualization of IL-22-expressing Lymphocytes Using Reporter Mice

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new transgenic reporter mouse model. We chose to visualize interleukin (IL) 22 gene expression because this cytokine has important activities in the intestine, where it contributes to repair tissues damaged by inflammation. Reporter systems offer considerable advantages over other methods of identifying products in vivo. In the case of IL-22, other studies had first isolated cells from tissues and then re-stimulated the cells in vitro. IL-22, which is normally secreted, was trapped inside cells using a drug, and intracellular staining was used to visualize it. This method identifies cells capable of producing IL-22, but it does not determine whether they were doing so in vivo. The reporter design includes inserting a gene for a fluorescent protein (tdTomato) into the IL-22 gene in such a way that the fluorescent protein cannot be secreted and therefore remains trapped inside the producing cells in vivo. Fluorescent producers can then be visualized in tissue sections or by ex vivo analysis through flow cytometry. The actual construction process for the reporter included recombineering a bacterial artificial chromosome that contained the IL-22 gene. This engineered chromosome was then introduced into the mouse genome. Homeostatic IL-22 reporter expression was observed in different mouse tissues, including the spleen, thymus, lymph nodes, Peyer&#39;s patch, and intestine, by flow cytometry analysis. Colitis was induced by T-cell (CD4+CD45RBhigh) transfer, and reporter expression was visualized. Positive T cells were first present in the mesenteric lymph nodes, and then they accumulated inside the lamina propria of the distal small intestine and colon tissues. The strategy using BACs gave good-fidelity reporter expression compared to IL-22 expression, and it is simpler than knock-in procedures.

We describe here a transgenic reporter mouse model to visualize the IL-22-producing cells inside different mouse tissues. This method can be used to track the location of other cytokines or secretary proteins in the mouse.

Reporter mice have been widely used to observe the localization of expression of targeted genes. This protocol focuses on a strategy to establish a new ...

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte extravasation might result in pathophysiological changes in the CNS. Thus, investigation of lymphocyte migration into the CNS is important to understand inflammatory CNS diseases and to develop new therapy approaches. Here we present an in vitro model of the human blood-brain barrier to study lymphocyte extravasation. Human brain microvascular endothelial cells (HBMEC) are confluently grown on a porous polyethylene terephthalate transwell insert to mimic the endothelium of the blood-brain barrier. Barrier function is validated by zonula occludens immunohistochemistry, transendothelial electrical resistance (TEER) measurements as well as analysis of evans blue permeation. This model allows investigation of the diapedesis of rare lymphocyte subsets such as CD56brightCD16dim/- NK cells. Furthermore, the effects of other cells, cytokines and chemokines, disease-related alterations, and distinct treatment regimens on the migratory capacity of lymphocytes can be studied. Finally, the impact of inflammatory stimuli as well as different treatment regimens on the endothelial barrier can be analyzed.

Analysis of Lymphocyte Extravasation Using an In Vitro Model of the Human Blood-brain Barrier

Here, we describe a human blood-brain barrier model enabling to investigate lymphocyte transmigration into the central nervous system in vitro.

Lymphocyte extravasation into the central nervous system (CNS) is critical for immune surveillance. Disease-related alterations of lymphocyte ...

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by fluorescence or bioluminescence. We utilize BlaC, an enzyme expressed constitutively by all M. tuberculosis strains. REF allows rapid quantification of bacteria in lungs of infected mice. The same group of mice can be imaged at many time points, greatly reducing costs, enumerating bacteria more quickly, allowing novel observations in host-pathogen interactions, and increasing statistical power, since more animals per group are readily maintained. REF is extremely sensitive due to the catalytic nature of the BlaC enzymatic reporter and specific due to the custom flourescence resonance energy transfer (FRET) or fluorogenic substrates used. REF does not require recombinant strains, ensuring normal host-pathogen interactions. We describe the imaging of M. tuberculosis infection using a FRET substrate with maximal emission at 800 nm. The wavelength of the substrate allows sensitive deep tissue imaging in mammals. We will outline aerosol infection of mice with M. tuberculosis, anesthesia of mice, administration of the REF substrate, and optical imaging. This method has been successfully applied to evaluating host-pathogen interactions and efficacy of antibiotics targeting M. tuberculosis.

Imaging Mycobacterium tuberculosis in Mice with Reporter Enzyme Fluorescence

We describe the optical imaging of mice infected with Mycobacterium tuberculosis (M. tuberculosis) using reporter enzyme fluorescence (REF). This protocol facilitates the sensitive and specific detection of M. tuberculosis in pre-clinical animal models for pathogenesis, therapeutics and vaccine research.

Reporter enzyme fluorescence (REF) utilizes substrates that are specific for enzymes present in target organisms of interest for imaging or detection by ...

CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we present a detailed protocol for the generation of novel in vitro models for HIV infection with chosen genomic integration sites using CRISPR-Cas9-based genome engineering technology. With this method, a reporter sequence of choice can be integrated into a targeted, chosen genomic locus, reflecting clinically relevant integration sites.
In the protocol, the design of an HIV-derived reporter and choosing of a target site and gRNA sequence are described. A targeting vector with homology arms is constructed and transfected into Jurkat T cells. The reporter sequence is targeted to the selected genomic site by homologous recombination facilitated by a Cas9-mediated double-strand break at the target site. Single-cell clones are generated and screened for targeting events by flow cytometry and PCR. Selected clones are then expanded, and correct targeting is verified by PCR, sequencing, and Southern blotting. Potential off-target events of CRISPR-Cas9-mediated genome engineering are analyzed.
By using this protocol, novel cell culture systems that model HIV infection at clinically relevant integration sites can be generated. Although the generation of single-cell clones and verification of correct reporter sequence integration is time-consuming, the resulting clonal lines are powerful tools to functionally analyze proviral integration site choice.

We present a genome engineering workflow for the generation of new in vitro models for HIV-1 infection that recapitulate proviral integration at selected genomic sites. Targeting of HIV-derived reporters is facilitated by CRISPR-Cas9-mediated, site-specific genome manipulation. Detailed protocols for single-cell clone generation, screening, and correct targeting verification are provided.

Human immunodeficiency virus (HIV) integrates its proviral DNA non-randomly into the host cell genome at recurrent sites and genomic hotspots. Here we ...

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of massage on skeletal muscles, the molecular mechanisms behind are poorly understood. We have recently developed a simple device to apply local cyclical compression (LCC), which can generate intramuscular pressure waves with varying amplitudes. Using this device, we have demonstrated that LCC modulates inflammatory responses of macrophages in situ and alleviates immobilization-induced muscle atrophy. Here, we describe protocols for the optimization and application of LCC as a massage-like intervention against immobilization-induced inflammation and atrophy of skeletal muscles of mouse hindlimbs. The protocol that we have developed can be useful for investigating the mechanism underlying beneficial effects of physical exercise and massage. Our experimental system provides a prototype of the analytical approach to elucidate the mechanical regulation of muscle homeostasis, although further development needs to be made for more comprehensive studies.

Here we describe the protocols for applying defined mechanical loads to mouse calves and for monitoring the concomitant intramuscular pressure changes. The experimental systems that we have developed can be useful for investigating the mechanism behind the beneficial effects of physical exercise and massage.

Massage is generally recognized to be beneficial for relieving pain and inflammation. Although previous studies have reported anti-inflammatory effects of ...

Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin injury, inflammation, and/or sensitization. The goal of this study is to describe an epidermal-dermal separation protocol using thermolysin, a proteinase that is active at 4 &deg;C. To illustrate this procedure, Sprague Dawley rats are anesthetized, and right hind paws are injected with carrageenan. Six and twelve hours after injection, rats with inflammation and na&iuml;ve rats are euthanized, and a piece of hind paw, glabrous skin is placed in cold Dulbecco's Modified Eagle Medium. The epidermis is then separated at the basement membrane from the dermis by thermolysin in PBS with calcium chloride. Next, the dermis is secured by microdissection forceps, and the epidermis is gently teased away. Toluidine blue staining of tissue sections show that the epidermis is separated cleanly from the dermis at the basement membrane. All keratinocyte cell layers remain intact, and the epidermal rete ridges along with indentations from dermal papillae are clearly observed. Qualitative and real-time RT-PCR is used to determine nerve growth factor and interleukin-6 expression levels. Western blotting and immunohistochemistry are finally performed to detect amounts of nerve growth factor. This report illustrates that cold thermolysin digestion is an effective method to separate epidermis from dermis for evaluation of mRNA and protein alterations during inflammation.

Presented here is a protocol for the separation of epidermis from dermis to evaluate inflammatory mediator production. Following inflammation, rat hind paw epidermis is separated from the dermis by thermolysin at 4 &#176;C. The epidermis is then used for mRNA analysis by RT-PCR and protein evaluation by western blot and immunohistochemistry.

Easy-to-use and inexpensive techniques are needed to determine the site-specific production of inflammatory mediators and neurotrophins during skin ...

Measurement of Specific Mycobacterial Mistranslation Rates with Gain-of-function Reporter Systems

Summary

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CRISPR-Cas9-based Genome Engineering to Generate Jurkat Reporter Models for HIV-1 Infection with Selected Proviral Integration Sites

Application of Consistent Massage-Like Perturbations on Mouse Calves and Monitoring the Resulting Intramuscular Pressure Changes

Separation of Rat Epidermis and Dermis with Thermolysin to Detect Site-Specific Inflammatory mRNA and Protein