The authors wish to thank Emilija Renke and Janet Yu for technical assistance. DCN is supported by grants from the United States Department of Defense (DR080205, DM102823, OR09055, and OR090059). RDH is supported by a grant from the Maryland Agriculture Experiment Station and an NIH Training Grant in Cell and Molecular Biology.

1. Determining Optimal Heating Conditions
First, one must experimentally determine the optimal incubation temperature and time to use for the heating step in the assay. For our PlyC model, it is important to note that E. coli co-transformed with plyCA and plyCB genes on separate expression plasmids has been shown to form a fully functional PlyC holoenzyme6. The 96 well microtiter plate preparation as well as cell growth conditions and the subsequent replica plating technique were adapted from examples provided in the literature12-16. An incubation time of 30 min will be suitable for this assay as results from previous thermal inactivation experiments dictate a rapid loss in activity during short-term incubation in environments exceeding physiological temperature (unpublished observation). The optimal incubation temperature for PlyC was elucidated by the following steps:
<ol>
 <li>Transform the expression construct pBAD24:plyCA (Ampr) into the competent E. coli strain DH5&alpha; pBAD33:plyCB (Cmr).</li>
 <li>Plate the transformants on a Luria-Bertani (LB) agar plate supplemented with ampicillin (100 &mu;g/ml) and chloramphenicol (35 &mu;g/ml). Incubate the plates overnight at 37 &deg;C.</li>
 <li>With a sterile toothpick, inoculate an individual colony into each row of wells (Figure 2a) in a sterile, clear, flat-bottomed 96 well, lidded microtiter plate that contains 200 &mu;l of LB supplemented with ampicillin and chloramphenicol.</li>
 <li>Securely place the 96 well microtiter plate on a 37 &deg;C shaking incubator and grow the bacteria overnight at 300 rpm.</li>
 <li>Retrieve the 96 well microtiter plate from the 37 &deg;C shaking incubator and replica plate to a new 96 well microtiter plate as follows:
 <ol class="lalpha">
 <li>Add 180 &mu;l of LB supplemented with ampicillin and chloramphenicol to each well of the replica plate.</li>
 <li>Transfer 20 &mu;l of bacteria from the original plate to the corresponding wells in the replica plate. This should result in an initial OD600 of ~0.5.</li>
 </ol>
 </li>
 <li>Securely place the replica plate on the 37 &deg;C shaking incubator and incubate the plate for 1 hr at 300 rpm, then add the inductant. In the case of the pBAD expression systems, the inductant is 0.25% arabinose. Other expression systems may require different inductants. Place the plate back on the 37 &deg;C shaking incubator and shake at 300 rpm for an additional 4 hr to allow for protein expression. Expression levels typically range between 10-20 &mu;g of PlyC.</li>
 <li>Once protein expression has concluded, retrieve the replica plate and pellet the bacteria by placing the 96 well microtiter plate in a refrigerated centrifuge that contains a swinging-bucket rotor that fits 96 well microtiter plates. Centrifuge at 3,000 rpm for 10 min at room temperature.</li>
 <li>Remove the media supernatant by slowly inverting the microtiter plate and gently decanting the media.</li>
 <li>Lyse the cells by adding 50 &mu;l of B-PER II Bacterial Protein Extraction Reagent to each well. Incubate the plate at room temperature for 20 min.</li>
 <li>Increase the volume in each well to 120 &mu;l by adding 70 &mu;l of phosphate buffered saline (PBS) pH 7.2.</li>
 <li>Pellet the insoluble cellular debris by spinning the plate at 3,000 rpm for 10 min at 4 &deg;C in the refrigerated centrifuge.</li>
 <li>Transfer 110 &mu;l of the soluble crude lysate to the corresponding wells of a 96 well thermocycler plate.</li>
 <li>Using 30 min as the predetermined incubation time, expose the soluble lysates currently residing in the 96 well thermocycler plate to a broad gradient temperature range spanning 15-20 &deg;C. Note, the goal is to determine at what temperature a particular enzyme loses &gt; 95% catalytic activity.</li>
 <li>After incubation, incubate the 96 well thermocycler plate at 4 &deg;C for 5 min and transfer 100 &mu;l of the soluble lysates from the 96 well thermocycler plate to their corresponding well location in the final 96 well microtiter plate.</li>
 <li>With a 12-channel multipipettor, add 100 &mu;l of GAS strain D471 to each well. Note, D471 cells are originally lyophilized from overnight cultures and resuspended with PBS pH 7.2 to obtain an OD600 of ~2.0.</li>
 <li>Immediately place the 96 well plate in the microplate spectrophotometer and monitor the enzyme kinetics by measuring the OD600 every 15 sec for 20 min. The lowest temperature incubation that corresponded to wells that lacked a change in optical density (&Delta;OD &le; 0.1) is defined as the non-permissive temperature.</li>
</ol>
After a broad temperature screen is performed to identify the non-permissive temperature, the above steps can be repeated over a more narrow range of temperatures (5 &deg;-10 &deg;C) to elucidate the precise non-permissive temperature for the enzyme of interest. Note, the non-permissive temperature identified in this assay may be different than the melting temperature elucidated by other means due to differences in volume, concentration, etc.
2. Generating the Mutant Library
The creation of the mutant library to be screened involves randomly incorporating mutations by an error-prone DNA polymerase with minimal nucleotide bias in the plyCA gene using the GeneMorph II Random Mutagenesis Kit as follows:
<ol>
 <li>Design nucleotide primers with similar melting temperatures (Tm) between 55-72 &deg;C with the restriction sites of choice at the 5' and 3' ends of the plyCA gene.</li>
 <li>Since the desired mutation rate is 2-3 nucleotides per plyCA (1.4 kb), use the PCR reaction component concentrations as well as the thermocycler conditions recommended by the manufacturer for low mutation frequencies (0-4.5 per kb).</li>
 <li>Clone the mutagenized plyCA genes into the expression vector pBAD24.</li>
 <li>Transform the constructs into a DH5&alpha; pBAD33:plyCB background and plate transformants on LB agar plates supplemented with ampicillin and chloramphenicol.</li>
 <li>Additionally, transform and plate DH5&alpha; pBAD33:plyCB with the expression vector pBAD24 containing the WT plyCA gene. Colonies from this plate will serve as the controls during screening.</li>
</ol>
3. 96 Well Microtiter Plate Preparation and Cell Growth Conditions
<ol>
 <li>Fill each well of a 96 well microtiter plate with 200 &mu;l of LB supplemented with ampicillin and chloramphenicol.</li>
 <li>Following the microtiter plate schematic (Figure 2b), carefully select an individual colony from the overnight agar plates with a sterilized toothpick and inoculate the bacteria in the designated well of the 96 well microtiter plate. It is essential to ensure only one colony per well is inoculated.</li>
 <li>Shake the securely anchored 96 well microtiter plate at 300 rpm overnight at 37 &deg;C.</li>
</ol>
4. Replica Plating, Protein Expression Induction and Lysate Preparation
<ol>
 <li>Follow steps 1.5-1.11 from the section titled "Determining the optimal heating conditions" with one modification. Store the original plate at 4 &deg;C after the replica plating step has concluded.</li>
 <li>After step 1.11, transfer 110 &mu;l of the soluble crude lysate to the corresponding wells of a 96 well thermocycler plate except for the positive control wells A1-D1. With regard to the positive control lysates (i.e. lysates that are not heated), transfer 100 &mu;l of the lysates to the corresponding wells in a new 96 well microtiter plate that will serve as the final assay plate for the experiment and store on ice.</li>
</ol>
5. Soluble Lysate Heat Treatment
<ol>
 <li>Heat the negative control and mutant soluble lysates currently confined in the 96 well thermocycler plate in the thermocycler for 30 min at the optimized non-permissive incubation temperature determined in step 1.</li>
 <li>After non-permissive temperature incubation, incubate the 96 well thermocycler plate at 4 &deg;C for 5 min.</li>
 <li>Transfer 100 &mu;l of the soluble lysates from the 96 well thermocycler plate to their identical well locations in the final 96 well microtiter assay plate already containing the positive control soluble lysates.</li>
 <li>With a 12-channel multipipettor, add 100 &mu;l of GAS strain D471 to each well. Immediately place the plate in the microplate spectrophotometer.</li>
 <li>Monitor the enzyme kinetics by measuring the OD600 every 15 sec for 20 min.
 <ol class="lalpha">
 <li>A PlyC variant with progressed thermal behavior is defined as a construct that can decrease the original optical density by 50 percent in less than 900 sec at the non-permissive temperature. Additionally, the positive controls (i.e. non-heated, WT PlyC) need to display WT catalytic activity (t1/2 &le; 100 sec) and the negative controls (i.e. heated, WT PlyC) should be devoid of activity.</li>
 </ol>
 </li>
 <li>Once a PlyC variant with progressed thermal behavior is identified, retrieve the original plate stored at 4 &deg;C and inoculate bacteria from the individual well specific to the mutant of interest into fresh LB media supplemented with ampicillin and chloramphenicol. Grow the inoculum overnight at 37 &deg;C.</li>
 <li>Extract and purify plasmid DNA from the culture and then submit for sequencing in order to identify the nucleotide mutations that infer enhanced thermal behavior to PlyCA. Additionally, supplement a small aliquot from the overnight culture with 10% glycerol and store at -80 &deg;C for further use.</li>
</ol>

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No conflicts of interest declared.

This protocol, outlined in Figure 1, presents a 96 well microtiter plate methodology that allows one to utilize directed evolution to increase the thermostability of any bacteriolytic enzyme. Through the use of an error-prone DNA polymerase, one can introduce random mutations that increase the overall kinetic stability of the translated bacteriolytic molecule of interest, which is typically due to molecular reorganizations consisting of increasing electrostatic, disulphide bridge and hydrophobic interactions that generate improved molecular packing, enhanced modifications of surface charge networks or reinforcement of a higher oligomerization state17-18. After the introduction of nucleotide mutations, an extensive screening procedure was then used to identify mutations that are thermally beneficial. The representative results presented here were based on one round of random mutagenesis. In reality, it has been suggested that at least three successive rounds of random mutagenesis, each using the most robust mutant as the lead enzyme, followed by DNA shuffling is appropriate for these types of directed evolution thermal behavior studies2.
It is important to understand that while this protocol identifies mutations that augment kinetic stability, these variants do not necessarily have increased thermostability. A molecule is considered thermostable when it has the ability to retain its structure at a high temperature. However, in the assay presented, the residual activity of the mutant lysates are not assayed at the same temperature that they were initially incubated at during the heat treatment step and thus one may be selecting for mutations that promote refolding rather than enhanced thermostability19. While we are extrapolating thermal behavior as an indication of thermal stability, true thermal stability must be measured empirically by biophysical methods such as circular dichroism (CD), differential scanning calorimetry (DSC) and differential scanning fluorimetry (DSF). Preliminary data from CD and DSF experiments suggest that several candidates identified from the methodology presented do indeed display progressed thermostability (data not shown).
Although the methodology presented here is specific to implementing increased thermal behavior to PlyC, this same methodology can additionally be employed to enhance thermal activity to other bacteriolytic enzymes with some minor alterations to variables such as buffers, mutation rates, heating conditions and expression systems. One critical variable associated with this assay relates to the nucleotide mutation rate of the error-prone DNA polymerase. We chose to use the error-prone Mutazyme II DNA polymerase in our directed evolution assay due to experimental evidence suggesting this particular enzyme displays the least amount of bias with regards to which nucleotides are randomly incorporated into the gene of interest when compared to other random mutagenesis techniques such as use of hydroxylamine, mutator E. coli strains and other error-prone DNA polyermases20. In general, lower mutation rates tend to be more desirable for two reasons. First, engineering thermostability to proteins typically consists of relatively few amino acid substitutions. High mutation rates can cause dramatic structural alterations to particular molecules that can conclusively result in structural misfolding and functional discrepancies. For example, the incorporation of glycine and proline residues can disrupt alpha helical secondary structures21. Second, high nucleotide mutation rates increase the chances of incorporating premature stop codons, resulting in truncated molecules that are biologically inactive. In general, lower mutation rates are preferred because lower error rates result in the accumulation of adaptive mutations while higher mutation rates generate neutral or deleterious mutations22.
For each round of random mutagenesis, another critical variable that one must optimize involves the incubation temperature used during the screening process. Selection of the experimental non-permissive temperature is relatively challenging. We initially used an incubation temperature that was 10 &deg;C higher than the non-permissive temperature of PlyC, however after screening 3,000 mutants, we were unable to identify any advantageous mutations. Keeping in mind directed evolution methodologies consist of sequentially identifying beneficial amino acid substitutions that have additive effects, it was determined that a less stringent temperature should be used during the screening process even if it increased the chance of generating false-positives. As such, we used an incubation temperature that was only a few degrees above the non-permissive temperature and rescreened selected candidates to rule out the false-positives.
We decided to utilize an incubation temperature that was not too temperate (&le; 1 &deg;C higher than lowest non-permissive temperature) and conversely not too harsh (&ge; 5 &deg;C higher than lowest non-permissive temperature). The ideal temperature we decided to use in this particular assay was a moderate 2 &deg;C higher than the experimentally determined non-permissive temperature. Choosing a temperature that is too mild will result in a much higher false positive identification rate than the ~20% we observed (Table I) when using a moderate incubation temperature. Furthermore, using an incubation temperature that is too harsh could ultimately result in the inability to identify mutants with significantly enhanced thermal behavior. For example, using an incubation temperature of &ge; 5 &deg;C would have resulted in the inability to identify the promising 29C3 mutant as well as the majority of the other candidates selected during the first round of screening.
In summation, we provide a protocol for engineering bacteriolytic enzymes to acquire enhanced kinetic stability. Moreover, this same assay, without the heating steps, can be used to screen for mutations that increase catalytic activity. Augmenting both catalytic activity and thermostability is an important developmental hurdle facing any therapeutic enzyme. While the details of this protocol are specific to the endolysin PlyC, the methodology can be adapted to any bacteriolytic enzyme with only a few alterations. We presented preliminary results from only the first round of mutagenesis that validates that functionality of the assay, resulting in the implementation and identification of mutations that generate an increase in molecular thermostability of significant magnitude.

<table border="1">
 <tbody>
 <tr>
 <td>Name of the reagent</td>
 <td>Company</td>
 <td>Catalogue number</td>
 </tr>
 <tr>
 <td>B-PER II Bacterial Protein Extraction Reagent</td>
 <td>Thermo Scientific</td>
 <td>78260</td>
 </tr>
 <tr>
 <td>GeneMorph II Random Mutagenesis Kit</td>
 <td>Agilent Technologies</td>
 <td>200500</td>
 </tr>
 <tr>
 <td>Clear 96 Well, Flat-bottomed Microtiter Plate With Lid</td>
 <td>BD Vacutainer Labware Medical</td>
 <td>353072</td>
 </tr>
 <tr>
 <td>96 Well Nonskirted PCR Plates</td>
 <td>Fisher Scientific </td>
 <td>14-230-232 </td>
 </tr>
 <tr>
 <td>Swing-bucket Rotor</td>
 <td>Eppendorf </td>
 <td>A-2-DWP </td>
 </tr>
 <tr>
 <td>Ampicillin Sodium Salt</td>
 <td>Fisher Scientific </td>
 <td>BP1760 </td>
 </tr>
 <tr>
 <td>Chloramphenicol </td>
 <td>Fisher Scientific </td>
 <td>BP904 </td>
 </tr>
 <tr>
 <td>Arabinose</td>
 <td>Fisher Scientific</td>
 <td>BP2504</td>
 </tr>
 <tr>
 <td>pBAD24 Expression Vector</td>
 <td>ATCC</td>
 <td>87399</td>
 </tr>
 <tr>
 <td>pBAD33 Expression Vector</td>
 <td>ATCC</td>
 <td>87402</td>
 </tr>
 </tbody>
</table>

a new screening method for the directed evolution of thermostable bacteriolytic enzymes

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not associated with the protein in nature. Literature has provided numerous examples regarding the implementation of directed evolution to successfully alter molecular specificity and catalysis1. The primary advantage of utilizing directed evolution instead of more rational-based approaches for molecular engineering relates to the volume and diversity of variants that can be screened2. One possible application of directed evolution involves improving structural stability of bacteriolytic enzymes, such as endolysins. Bacteriophage encode and express endolysins to hydrolyze a critical covalent bond in the peptidoglycan (i.e. cell wall) of bacteria, resulting in host cell lysis and liberation of progeny virions. Notably, these enzymes possess the ability to extrinsically induce lysis to susceptible bacteria in the absence of phage and furthermore have been validated both in vitro and in vivo for their therapeutic potential3-5. The subject of our directed evolution study involves the PlyC endolysin, which is composed of PlyCA and PlyCB subunits6. When purified and added extrinsically, the PlyC holoenzyme lyses group A streptococci (GAS) as well as other streptococcal groups in a matter of seconds and furthermore has been validated in vivo against GAS7. Significantly, monitoring residual enzyme kinetics after elevated temperature incubation provides distinct evidence that PlyC loses lytic activity abruptly at 45 &deg;C, suggesting a short therapeutic shelf life, which may limit additional development of this enzyme. Further studies reveal the lack of thermal stability is only observed for the PlyCA subunit, whereas the PlyCB subunit is stable up to ~90 &deg;C (unpublished observation). In addition to PlyC, there are several examples in literature that describe the thermolabile nature of endolysins. For example, the Staphylococcus aureus endolysin LysK and Streptococcus pneumoniae endolysins Cpl-1 and Pal lose activity spontaneously at 42 &deg;C, 43.5 &deg;C and 50.2 &deg;C, respectively8-10. According to the Arrhenius equation, which relates the rate of a chemical reaction to the temperature present in the particular system, an increase in thermostability will correlate with an increase in shelf life expectancy11. Toward this end, directed evolution has been shown to be a useful tool for altering the thermal activity of various molecules in nature, but never has this particular technology been exploited successfully for the study of bacteriolytic enzymes. Likewise, successful accounts of progressing the structural stability of this particular class of antimicrobials altogether are nonexistent. In this video, we employ a novel methodology that uses an error-prone DNA polymerase followed by an optimized screening process using a 96 well microtiter plate format to identify mutations to the PlyCA subunit of the PlyC streptococcal endolysin that correlate to an increase in enzyme kinetic stability (Figure 1). Results after just one round of random mutagenesis suggest the methodology is generating PlyC variants that retain more than twice the residual activity when compared to wild-type (WT) PlyC after elevated temperature treatment.

At the conclusion of the first round of random mutagenesis, over 6,000 PlyC mutants were screened and a total of 35 mutants with potentially increased thermal behavior were identified, selected and sequenced. Genomic analysis, summarized in Table I, suggests that of the 35 candidates, 7 of the constructs contained WT PlyCA sequences at the level of translation, corresponding to false positives identified by the assay. Of the remaining 28 candidates, the mutation range was from 1 to 6 nucleotide mutations with an average mutation rate of 2.75 nucleotides per plyCA gene, which was in the 2-3 nucleotide mutation range we were targeting. At the translational level, this particular nucleotide mutation range and frequency yielded an amino acid mutation range of 1 to 5 amino acids, with an average mutation rate of 1.9 amino acid mutations per PlyCA polypeptide.
Of the 28 candidates with at least one amino acid mutation, four of these mutant constructs were randomly chosen for further characterization to validate that the extensive screening process of the directed evolution methodology was indeed functioning properly. The mutant PlyC enzymes were purified to &gt; 95% homogeneity based on SDS-PAGE analysis as previously described6-7. Enzyme kinetics of WT PlyC and each of the four PlyC mutants were characterized at equal molar concentrations after incubating the purified enzymes at various elevated temperatures. Activity was monitored after the addition of D471 GAS by measuring the optical density at 600 nm every 15 sec for 20 min. Activity was defined as the residual maximum velocity of the enzyme after heat incubation. Of the four candidates randomly selected for further characterization, mutant 29C3 showed the most enhanced thermal behavior. The thermal behavior of WT PlyC and 29C3 was investigated at different temperatures while incubating and assaying for activity in PBS pH 7.2 at 80 nM and 40 nM concentrations, respectively. The 45-50 &deg;C incubation experiments (Figure 3) were performed in a thermocycler where the samples were incubated in a thin-walled 96 well thermocycler plate in a total volume of 120 &mu;l. The 35 &deg;C, 40 &deg;C and 45 &deg;C incubation experiments (Figure 4 and 5) were performed in a heat block where the samples were incubated in a 1.5 ml microcentrifuge tube in a total volume of 1.3 ml. All experiments were performed in triplicates.
WT PlyC and 29C3 showed no significant difference in kinetic stability at room temperature (25 &deg;C) as depicted in the first set of bars in Figure 3. However, after a short-term incubation for 30 min from temperatures ranging from 45-50 &deg;C, the activity of 29C3 was substantially greater than the activity specific to the WT construct at each temperature point. For example, WT PlyC displayed a 44% loss in activity at 45.2 &deg;C whereas 29C3 showed only a 2% loss in activity at the same temperature.
Long-term incubation studies comparing the residual activity of both WT PlyC and 29C3 were additionally performed at 35 &deg;C and 40 &deg;C involving the measurement of residual activity at 24 and 48 hr time points. At 35 &deg;C, 29C3 displayed 41% and 176% higher activity than WT PlyC at 24 and 48 hr incubation time points, respectively (Figure 4a). At 40 &deg;C, 29C3 displayed 28% and 107% higher activity than WT PlyC at 24 and 48 hr incubation time points, respectively (Figure 4b).
The residual activity of WT PlyC and 29C3 were also monitored every 20 min for a total of 3 hr at 45 &deg;C. WT PlyC was only able to retain 21% activity after a 3 hr incubation at this temperature whereas 29C3 was able to retain 46% activity. The half-life (t1/2) for WT PlyC and 29C3 were 67 and 147 min, respectively, suggesting that 29C3 has a 2.2 fold increase in kinetic stability at 45 &deg;C (Figure 5).
<table border="1">
 <tbody>
 <tr>
 <td>Total Round 1 Candidates</td>
 <td>35</td>
 </tr>
 <tr>
 <td>Candidates with WT PlyCA Sequence</td>
 <td>7</td>
 </tr>
 <tr>
 <td>Candidates with &ge;1 Amino Acid Mutation</td>
 <td>28</td>
 </tr>
 <tr>
 <td>&mdash; Average Nucleotide Mutation Rate (nt/plyCA)</td>
 <td>2.75</td>
 </tr>
 <tr>
 <td>&mdash; Nucleotide Mutation Range (nt)</td>
 <td>1-6</td>
 </tr>
 <tr>
 <td>&mdash; Average Amino Acid Mutation Rate (AA/PlyCA)</td>
 <td>1.9</td>
 </tr>
 <tr>
 <td>&mdash; Amino Acid Mutation Range (AA)</td>
 <td>1-5</td>
 </tr>
 </tbody>
</table>
Table 1. Candidate Pool Genomic Analysis.
<img alt="Figure 1" src="/files/ftp_upload/4216/4216fig1.jpg" fo:content-width="5.5in" fo:src="/files/ftp_upload/4216/4216fig1highres.jpg"/> 
Figure 1. Directed evolution assay methodology. In directed evolution, one starts with the lead enzyme, which would be WT PlyC for the first round. A library of PlyC mutants containing random unbiased nucleotide mutations to the plyCA gene is then constructed by an error-prone DNA polymerase, cloned into the expression vector pBAD24 and transformed into the E. coli strain DH5&alpha; already containing pBAD33:plyCB. Individual transformants are inoculated into their own specific well of a 96 well microtiter plate. Through an extensive screening process, individual PlyC mutants that are catalytically active after incubation at a non-permissible temperature are classified as mutants with enhanced kinetic stability. Theconstruct displaying the most progressed thermal behavior will consequently becomes the lead enzyme for the next round of random mutagenesis. Overall, there are three complete rounds of random mutagenesis followed by DNA shuffling resulting in a bacteriolytic molecule with evolved thermal behavior. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/4216/4216fig1large.jpg" target="_blank"> Click here to view larger figure</a>.
<img alt="Figure 2" src="/files/ftp_upload/4216/4216fig2.jpg" /> 
Figure 2. 96 well microtiter plate templates for directed evolution assay. The microtiter plate schematic during the determination of the optimal heating conditions (Figure 2a) consists of inoculating a single parental WT PlyC clone into each row of the microtiter plate. The microtiter plate schematic during mutant screening (Figure 2b) consists of inoculating each well in column 1 with a unique parental WT PlyC clone as well as inoculating each well in columns 2-12 with a distinct PlyC mutant clone. Wells A1-D1 are designated for the positive parental controls, which consist of WT PlyC constructs not exposed to non-permissible temperature incubation. Wells E1-H1 are designated for the negative parental controls, which consist of WT PlyC constructs that are exposed to non-permissible temperature incubation.
<img alt="Figure 3" src="/files/ftp_upload/4216/4216fig3.jpg" /> 
Figure 3. Residual activity kinetic analysis comparing WT PlyC and 29C3. Enzymes were purified to homogeneity and incubated for 30 min in a thermocycler at equal molar concentrations at either room temperature or at a temperature gradient ranging from 45-50 &deg;C. Enzymatic activity correlates with the maximum velocity displayed after specific temperature incubation. With the exception of 25 &deg;C and 47.7 &deg;C, the variation in activity between WT and 29C3 at each temperature correlated to a p-value &lt; 0.05. All data are reported as the mean &plusmn;SEM of three independent experiments.
<img alt="Figure 4" src="/files/ftp_upload/4216/4216fig4.jpg" /> 
Figure 4. Residual activity kinetic analysis comparing WT PlyC to 29C3 at 35 &deg;C and 40 &deg;C. Equal molar concentrations of purified WT PlyC and 29C3 were incubated in a heat block at 35 &deg;C (Figure 4a) or 40 &deg;C (Figure 4b). The residual enzyme activity was measured at 24 and 48 hr time points. The activity displayed by each construct was normalized to the maximum velocity displayed at time point zero. The variation in activity between WT and 29C3 at each temperature and time point correlated to a p-value &lt; 0.05. All data are reported as the mean &plusmn;SEM of three independent experiments.
<img alt="Figure 5" src="/files/ftp_upload/4216/4216fig5.jpg" /> 
Figure 5. Residual activity kinetic analysis comparing WT PlyC to 29C3 at 45 &deg;C. Equal molar concentrations of purified WT PlyC and 29C3 were incubated in a heat block at 45&deg;C and the residual enzyme activity was measured every 20 min for 3 hr. The activity displayed by each construct was normalized to the maximum velocity displayed at time point zero. With the exception of the data points at 20 min, the variation in activity between WT and 29C3 at each time point correlated to a p-value &lt; 0.05. All data are reported as the mean &plusmn;SEM of three independent experiments.

Watch this Scientific Journal Video about A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes at JoVE.com

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A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

Directed evolution is defined as a method to harness natural selection in order to engineer proteins to acquire particular properties that are not ...

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17.8K Views.  University of Maryland. A novel directed evolution method specific to the field of thermostability engineering was developed and consequently validated for bacteriolytic enzymes. After only one round of random mutagenesis, an evolved bacteriolytic enzyme, PlyC 29C3, displayed greater than twice the residual activity when compared to the wild-type protein after elevated temperature incubation.

Video: A New Screening Method for the Directed Evolution of Thermostable Bacteriolytic Enzymes

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the disease, is characterised by an accumulation of Plasmodium falciparum infected red blood cells (iRBC) at pigmented trophozoite stage in the microvasculature of the brain2-4. This microvessel obstruction (sequestration) leads to acidosis, hypoxia and harmful inflammatory cytokines (reviewed in 5). Sequestration is also found in most microvascular tissues of the human body2, 3. The mechanism by which iRBC attach to the blood vessel walls is still poorly understood.
The immortalized Human Brain microvascular Endothelial Cell line (HBEC-5i) has been used as an in vitro model of the blood-brain barrier6. However, Plasmodium falciparum iRBC attach only poorly to HBEC-5i in vitro, unlike the dense sequestration that occurs in cerebral malaria cases. We therefore developed a panning assay to select (enrich) various P. falciparum strains for adhesion to HBEC-5i in order to obtain populations of high-binding parasites, more representative of what occurs in vivo.
A sample of a parasite culture (mixture of iRBC and uninfected RBC) at the pigmented trophozoite stage is washed and incubated on a layer of HBEC-5i grown on a Petri dish. After incubation, the dish is gently washed free from uRBC and unbound iRBC. Fresh uRBC are added to the few iRBC attached to HBEC-5i and incubated overnight. As schizont stage parasites burst, merozoites reinvade RBC and these ring stage parasites are harvested the following day. Parasites are cultured until enough material is obtained (typically 2 to 4 weeks) and a new round of selection can be performed. Depending on the P. falciparum strain, 4 to 7 rounds of selection are needed in order to get a population where most parasites bind to HBEC-5i. The binding phenotype is progressively lost after a few weeks, indicating a switch in variant surface antigen gene expression, thus regular selection on HBEC-5i is required to maintain the phenotype.
In summary, we developed a selection assay rendering P. falciparum parasites a more "cerebral malaria adhesive" phenotype. We were able to select 3 out of 4 P. falciparum strains on HBEC-5i. This assay has also successfully been used to select parasites for binding to human dermal and pulmonary endothelial cells. Importantly, this method can be used to select tissue-specific parasite populations in order to identify candidate parasite ligands for binding to brain endothelium. Moreover, this assay can be used to screen for putative anti-sequestration drugs7.

Selection of Plasmodium falciparum Parasites for Cytoadhesion to Human Brain Endothelial Cells

An in vitro model for cerebral malaria sequestration is described1. Plasmodium falciparum infected red blood cells are selected for binding to immortalized human brain microvascular endothelial cells. The selected parasites show a distinct phenotype. The selection process can be applied using various P. falciparum strains and endothelial cell lines.

Most human malaria deaths are caused by blood-stage Plasmodium falciparum parasites. Cerebral malaria, the most life-threatening complication of the ...

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. 1A-C), females of the genus Leptopilina (Fig. 1D, 1F, 1G) and Ganaspis (Fig. 1E) attack the larval stages. We use these parasites to study the molecular basis of a biological arms race. Parasitic wasps have tremendous value as biocontrol agents. Most of them carry virulence and other factors that modify host physiology and immunity. Analysis of Drosophila wasps is providing insights into how species-specific interactions shape the genetic structures of natural communities. These studies also serve as a model for understanding the hosts' immune physiology and how coordinated immune reactions are thwarted by this class of parasites.
The larval/pupal cuticle serves as the first line of defense. The wasp ovipositor is a sharp needle-like structure that efficiently delivers eggs into the host hemocoel. Oviposition is followed by a wound healing reaction at the cuticle (Fig. 1C, arrowheads). Some wasps can insert two or more eggs into the same host, although the development of only one egg succeeds. Supernumerary eggs or developing larvae are eliminated by a process that is not yet understood. These wasps are therefore referred to as solitary parasitoids.
Depending on the fly strain and the wasp species, the wasp egg has one of two fates. It is either encapsulated, so that its development is blocked (host emerges; Fig. 2 left); or the wasp egg hatches, develops, molts, and grows into an adult (wasp emerges; Fig. 2 right). L. heterotoma is one of the best-studied species of Drosophila parasitic wasps. It is a "generalist," which means that it can utilize most Drosophila species as hosts1. L. heterotoma and L. victoriae are sister species and they produce virus-like particles that actively interfere with the encapsulation response2. Unlike L. heterotoma, L. boulardi is a specialist parasite and the range of Drosophila species it utilizes is relatively limited1. Strains of L. boulardi also produce virus-like particles3 although they differ significantly in their ability to succeed on D. melanogaster1. Some of these L. boulardi strains are difficult to grow on D. melanogaster1 as the fly host frequently succeeds in encapsulating their eggs. Thus, it is important to have the knowledge of both partners in specific experimental protocols.
In addition to barrier tissues (cuticle, gut and trachea), Drosophila larvae have systemic cellular and humoral immune responses that arise from functions of blood cells and the fat body, respectively. Oviposition by L. boulardi activates both immune arms1,4. Blood cells are found in circulation, in sessile populations under the segmented cuticle, and in the lymph gland. The lymph gland is a small hematopoietic organ on the dorsal side of the larva. Clusters of hematopoietic cells, called lobes, are arranged segmentally in pairs along the dorsal vessel that runs along the anterior-posterior axis of the animal (Fig. 3A). The fat body is a large multifunctional organ (Fig. 3B). It secretes antimicrobial peptides in response to microbial and metazoan infections.
Wasp infection activates immune signaling (Fig. 4)4. At the cellular level, it triggers division and differentiation of blood cells. In self defense, aggregates and capsules develop in the hemocoel of infected animals (Fig. 5)5,6. Activated blood cells migrate toward the wasp egg (or wasp larva) and begin to form a capsule around it (Fig. 5A-F). Some blood cells aggregate to form nodules (Fig. 5G-H). Careful analysis reveals that wasp infection induces the anterior-most lymph gland lobes to disperse at their peripheries (Fig. 6C, D).
We present representative data with Toll signal transduction pathway components Dorsal and Sp&auml;tzle (Figs. 4,5,7), and its target Drosomycin (Fig. 6), to illustrate how specific changes in the lymph gland and hemocoel can be studied after wasp infection. The dissection protocols described here also yield the wasp eggs (or developing stages of wasps) from the host hemolymph (Fig. 8).

An Introduction to Parasitic Wasps of Drosophila and the Antiparasite Immune Response

Parasitoid (parasitic) wasps constitute a major class of natural enemies of many insects including Drosophila melanogaster. We will introduce the techniques to propagate these parasites in Drosophila spp. and demonstrate how to analyze their effects on immune tissues of Drosophila larvae.

Most known parasitoid wasp species attack the larval or pupal stages of Drosophila. While Trichopria drosophilae infect the pupal stages of the host (Fig. ...

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

A Primary Neuron Culture System for the Study of Herpes Simplex Virus Latency and Reactivation

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent reactivation events that ensure transmission and contribute to clinical disease. Current antivirals do not impact the latent reservoir and there are no vaccines. While the molecular details of lytic replication are well-characterized, mechanisms controlling latency in neurons remain elusive. Our present understanding of latency is derived from in vivo studies using small animal models, which have been indispensable for defining viral gene requirements and the role of immune responses. However, it is impossible to distinguish specific effects on the virus-neuron relationship from more general consequences of infection mediated by immune or non-neuronal support cells in live animals. In addition, animal experimentation is costly, time-consuming, and limited in terms of available options for manipulating host processes. To overcome these limitations, a neuron-only system is desperately needed that reproduces the in vivo characteristics of latency and reactivation but offers the benefits of tissue culture in terms of homogeneity and accessibility.
Here we present an in vitro model utilizing cultured primary sympathetic neurons from rat superior cervical ganglia (SCG) (Figure 1) to study HSV-1 latency and reactivation that fits most if not all of the desired criteria. After eliminating non-neuronal cells, near-homogeneous TrkA+ neuron cultures are infected with HSV-1 in the presence of acyclovir (ACV) to suppress lytic replication. Following ACV removal, non-productive HSV-1 infections that faithfully exhibit accepted hallmarks of latency are efficiently established. Notably, lytic mRNAs, proteins, and infectious virus become undetectable, even in the absence of selection, but latency-associated transcript (LAT) expression persists in neuronal nuclei. Viral genomes are maintained at an average copy number of 25 per neuron and can be induced to productively replicate by interfering with PI3-Kinase / Akt signaling or the simple withdrawal of nerve growth factor1. A recombinant HSV-1 encoding EGFP fused to the viral lytic protein Us11 provides a functional, real-time marker for replication resulting from reactivation that is readily quantified. In addition to chemical treatments, genetic methodologies such as RNA-interference or gene delivery via lentiviral vectors can be successfully applied to the system permitting mechanistic studies that are very difficult, if not impossible, in animals. In summary, the SCG-based HSV-1 latency / reactivation system provides a powerful, necessary tool to unravel the molecular mechanisms controlling HSV1 latency and reactivation in neurons, a long standing puzzle in virology whose solution may offer fresh insights into developing new therapies that target the latent herpesvirus reservoir.

The protocol describes an efficient and reproducible model system to study herpes simplex virus type 1 (HSV-1) latency and reactivation. The assay employs homogenous sympathetic neuron cultures and allows for the molecular dissection of virus-neuron interactions using a variety of tools including RNA interference and expression of recombinant proteins.

Herpes simplex virus type-1 (HSV-1) establishes a life-long latent infection in peripheral neurons. This latent reservoir is the source of recurrent ...

Assay of Adhesion Under Shear Stress for the Study of T Lymphocyte-Adhesion Molecule Interactions

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and interaction with antigen presenting cells (APC) in the formation of immunological synapses. These two contexts of T cell adhesion differ in that T cell-APC interactions can be considered static, while T cell-blood vessel interactions are challenged by the shear stress generated by circulation itself. T cell-APC interactions are classified as static in that the two cellular partners are static relative to each other. Usually, this interaction occurs within the lymph nodes. As a T cell interacts with the blood vessel wall, the cells arrest and must resist the generated shear stress.1,2 These differences highlight the need to better understand static adhesion and adhesion under flow conditions as two distinct regulatory processes. The regulation of T cell adhesion can be most succinctly described as controlling the affinity state of integrin molecules expressed on the cell surface, and thereby regulating the interaction of integrins with the adhesion molecule ligands expressed on the surface of the interacting cell. Our current understanding of the regulation of integrin affinity states comes from often simplistic in vitro model systems. The assay of adhesion using flow conditions described here allows for the visualization and accurate quantification of T cell-epithelial cell interactions in real time following a stimulus. An adhesion under flow assay can be applied to studies of adhesion signaling within T cells following treatment with inhibitory or stimulatory substances. Additionally, this assay can be expanded beyond T cell signaling to any adhesive leukocyte population and any integrin-adhesion molecule pair.

This flow adhesion assay provides a simple, high impact model of T cell-epithelial cell interactions. A syringe pump is used to generate shear stress, and confocal microscopy captures images for quantification. The goal of these studies is to effectively quantify T cell adhesion using flow conditions.

Overall, T cell adhesion is a critical component of function, contributing to the distinct processes of cellular recruitment to sites of inflammation and ...

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella spp./Shigella spp. The gold standard method for the detection of Salmonella spp./Shigella spp. is direct culture but this is labor-intensive and time-consuming. Here, we describe a high-throughput platform for Salmonella spp./Shigella spp. screening, using real-time polymerase chain reaction (PCR) combined with guided culture. There are two major stages: real-time PCR and the guided culture. For the first stage (real-time PCR), we explain each step of the method: sample collection, pre-enrichment, DNA extraction and real-time PCR. If the real-time PCR result is positive, then the second stage (guided culture) is performed: selective culture, biochemical identification and serological characterization. We also illustrate representative results generated from it. The protocol described here would be a valuable platform for the rapid, specific, sensitive and high-throughput screening of Salmonella spp./Shigella spp.

A High-throughput Platform for the Screening of Salmonella spp./Shigella spp.

Salmonella spp./Shigella spp. are common pathogens attributed to diarrhea. Here, we describe a high-throughput platform for the screening of Salmonella spp./Shigella spp. using real-time PCR combined with guided culture.

Fecal-oral transmission of acute gastroenteritis occurs from time to time, especially when people who handled food and water are infected by Salmonella ...

Arbovirus Infections As Screening Tools for the Identification of Viral Immunomodulators and Host Antiviral Factors

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. However, these screens are typically conducted in cells that are naturally permissive to the viral pathogen under study. Therefore, the robust replication of viruses in control conditions may limit the dynamic range of these screens. Furthermore, these screens may be unable to easily identify cellular defense pathways that restrict virus replication if the virus is well-adapted to the host and capable of countering antiviral defenses. In this article, we describe a new paradigm for exploring virus-host interactions through the use of screens that center on naturally abortive infections by arboviruses such as vesicular stomatitis virus (VSV). Despite the ability of VSV to replicate in a wide range of dipteran insect and mammalian hosts, VSV undergoes a post-entry, abortive infection in a variety of cell lines derived from lepidopteran insects, such as the gypsy moth (Lymantria dispar). However, these abortive VSV infections can be &#34;rescued&#34; when host cell antiviral defenses are compromised. We describe how VSV strains encoding convenient reporter genes and restrictive L. dispar cell lines can be paired to set-up screens to identify host factors involved in arbovirus restriction. Furthermore, we also show the utility of these screening tools in the identification of virally encoded factors that rescue VSV replication during coinfection or through ectopic expression, including those encoded by mammalian viruses. The natural restriction of VSV replication in L. dispar cells provides a high signal-to-noise ratio when screening for the conditions that promote VSV rescue, thus enabling the use of simplistic luminescence- and fluorescence-based assays to monitor the changes in VSV replication. These methodologies are valuable for understanding the interplay between host antiviral responses and viral immune evasion factors.

Here, we present the protocols to identify 1) virus-encoded immunomodulators that promote arbovirus replication and 2) eukaryotic host factors that restrict arbovirus replication. These fluorescence- and luminescence-based methods allow researchers to rapidly obtain quantitative readouts of arbovirus replication in simplistic assays with low signal-to-noise ratios.

RNA interference- and genome editing-based screening platforms have been widely used to identify host cell factors that restrict virus replication. ...

Co-immunoprecipitation Assay for Studying Functional Interactions Between Receptors and Enzymes

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with cytoplasmic tails of the receptors. The interactions often occur through specific protein domains and result in activation of the enzymes. There are several methods to study interactions between proteins. While co-immunoprecipitation is commonly used to study domains that are required for protein-protein interactions, there are no assays that document the contribution of specific domains to activity of the recruited enzymes at the same time. Accordingly, the method described here combines co-immunoprecipitation and an on-bead enzymatic activity assay for simultaneous evaluation of interactions between proteins and associated enzymatic activation. The goal of this protocol is to identify the domains that are critical for physical interactions between a protein and enzyme and the domains that are obligatory for complete activation of the enzyme. The importance of this assay is demonstrated, as certain receptor protein domains contribute to the binding of the enzyme to the cytoplasmic tail of the receptor, while other domains are necessary to regulate the function of the same enzyme.

Here, we present a protocol for co-immunoprecipitation and an on-bead enzymatic activity assay to simultaneously study the contribution of specific protein domains of plasma membrane receptors to both enzyme recruitment and enzyme activity.

Receptor-associated enzymes are the major mediators of cellular activation. These enzymes are regulated, at least in part, by physical interactions with ...

Bacterial Peptide Display for the Selection of Novel Biotinylating Enzymes

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic biotinylation by the E. coli biotin-protein ligase BirA is highly specific and allows for the biotinylation of target proteins in their native environment; however, the current usage of BirA mediated biotinylation requires the presence of a synthetic acceptor peptide (AP) in the target protein. Therefore, its application is limited to proteins that have been engineered to contain the AP. The purpose of the present protocol is to use the bacterial display of a peptide derived from an unmodified target protein to select for BirA variants that biotinylates the peptide. The system is based on a single plasmid that allows for the co-expression of BirA variants along with a scaffold for the peptide display on the bacterial surface. The protocol describes a detailed procedure for the incorporation of the target peptide into the display scaffold, creation of the BirA library, selection of active BirA variants and initial characterization of the isolated BirA variants. The method provides a highly effective selection system for the isolation of novel BirA variants that can be used for the further directed evolution of biotin-protein ligases that biotinylate a native protein in complex solutions.

Here we present a method to select for novel variants of the E. coli biotin-protein ligase BirA that biotinylates a specific target peptide. The protocol describes the construction of a plasmid for the bacterial display of the target peptide, generation of a BirA library, selection and characterization of BirA variants.

Biotin is an attractive post-translational modification of proteins that provides a powerful tag for the isolation and detection of protein. Enzymatic ...