Difference between revisions of "Part:BBa K5087002"

 
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    <img src="https://static.igem.wiki/teams/5087/part-registry-images/prymdetect-toolkit.png" alt="Part of the PrymDetect Toolkit" style="width:140px; float:left; margin-right:1em;">
  
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<!-- Add more about the biology of this part here
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    <h1>Introduction</h1>
−
===Usage and Biology===
+
    <p>ModF is a forward primer specifically designed to amplify a fragment of <b><i>Prymnesium parvum</i></b> genomic DNA in an <b>RPA</b> (Recombinase Polymerase Amplification) reaction by targeting the <b>ITS2</b> region in the genome.</p>
 +
    <p>This primer contains a <b>T7 promoter</b> to enable <i>in vitro transcription</i> of the amplified product into RNA, preparing it for <b>SHERLOCK</b> detection with Cas13 proteins.</p>
  
 +
    <h1>Biology and Usage</h1>
 +
    <h2>ITS Sequences</h2>
 +
    <p>The ITS2 (Internal Transcribed Spacer 2) region is a non-coding segment of DNA found within the <b>ribosomal RNA (rRNA)</b> gene cluster. In the genome of <i>Prymnesium parvum</i>, the ITS2 region lies between the <b>5.8S</b> and <b>nuclear large rRNA genes</b> [1].</p>
 +
    <p>The ITS regions, including ITS2, are commonly used for species identification because they tend to vary between species while being conserved within a species [2]. This variability makes the ITS2 region an effective target for designing <b>species-specific primers</b>, such as those used to identify <i>Prymnesium parvum</i> [3].</p>
 +
 +
    <h2>RPA Reaction</h2>
 +
    <p><b>Recombinase Polymerase Amplification (RPA)</b> is an isothermal nucleic acid amplification technique that operates at a temperature range of <b>37–42°C</b>, distinguishing it from traditional PCR methods that require thermal cycling for denaturation and annealing of DNA [4].</p>
 +
    <p>RPA relies on three essential types of proteins: <b>a recombinase</b>, <b>single-stranded DNA binding proteins (SSBs)</b>, and <b>a strand-displacing DNA polymerase</b>.</p>
 +
    <p>The process begins when the <b>recombinase protein</b> binds to a primer (about 30–35 nucleotides long) that matches the target DNA sequence. This complex then searches for homologous sequences in double-stranded DNA and initiates strand invasion. The <b>SSBs</b> stabilize the displaced strand to prevent primer dissociation, while the <b>strand-displacing DNA polymerase</b> extends the primer, resulting in exponential amplification of the target sequence [5].</p>
 +
 +
    <center>
 +
        <img src="https://static.igem.wiki/teams/5087/part-registry-images/rpa.png" alt="RPA Mechanism" style="width:100%;max-width:250px;">
 +
    </center>
 +
    <center>
 +
        <b>Figure 1.</b> The RPA <i>(Recombinase Polymerase Amplification)</i> Mechanism
 +
    </center>
 +
 +
    <h2>SHERLOCK Method</h2>
 +
    <p>The <b>SHERLOCK</b> platform is a modern synthetic biology tool that utilizes the properties of the <b>Cas13a</b> protein, an enzyme from the Nobel Prize-winning <b>CRISPR-Cas</b> system. The <b>Cas13a</b> protein is guided with high specificity to the target sequence using <b>crRNA</b>.</p>
 +
 +
    <p>The <b>crRNA</b> molecule is crucial for the assay's specificity. It consists of a <b>direct repeat (DR)</b> sequence and a <b>spacer sequence</b> that is complementary to the target. The <b>crRNA</b> molecule is designed to uniquely identify the organism by targeting the <b>Internal Transcribed Spacer (ITS)</b> sequence in its genome.</p>
 +
 +
    <p>First, the <b>Cas13a</b> protein binds to the organism's genetic material, which was previously amplified using RPA and transcribed into RNA. Once bound, the <b>Cas13a</b> protein is activated and exhibits a “collateral” <b>RNase</b> activity, meaning it non-specifically cleaves nearby single-stranded RNA molecules [7].</p>
 +
 +
    <p>This activity can be used in assays by including synthetic RNA probes tagged with a <b>fluorescent reporter</b> and a <b>quencher</b> in the reaction mixture. A fluorescent signal indicates that the reporters have been cleaved by <b>Cas13</b>, confirming the presence of the DNA target in the sample. The SHERLOCK method can also be used with Lateral Flow Assays (LFA). </p>
 +
 +
    <center>
 +
        <img src="https://static.igem.wiki/teams/5087/part-registry-images/sherlock.png" alt="SHERLOCK Method" style="width:100%;max-width:500px;">
 +
    </center>
 +
    <center>
 +
        <b>Figure 2.</b> The SHERLOCK <i>(Specific High-sensitivity Enzymatic Reporter unLOCKing)</i> Method Mechanism
 +
    </center>
 +
 +
 +
    <h1>Part Performance</h1>
 +
    <p>This part is an integral component of the PrymDetect Toolkit, which is designed for detecting <i>Prymnesium parvum</i> in water samples using the SHERLOCK method. Our team evaluated this part alongside other components of the toolkit to determine the most reliable configurations and make its use easier for future iGEM teams.</p>
 +
   
 +
    <center>
 +
        <img src="https://static.igem.wiki/teams/5087/part-registry-images/modf.png" alt="Fluorescence Readout" style="width:100%;max-width:1000px;">
 +
    </center>
 +
    <center>
 +
        <b>Figure 3:</b> Fluorescence readout results demonstrating the performance of various part combinations from our toolkit, with the ModF primer serving as the connecting component. For simplicity, the negative controls for both crRNAs are not shown, as they were all negative as anticipated.
 +
    </center>
 +
 +
    <h2>Parts Compatible with ModF</h2>
 +
    <p>The results show that <b>ModF</b> performs best when paired with the <b>GalR</b> primer and the <b>PrymCrRNA1</b> design. This combination delivers the highest fluorescence readout intensity and provides results the fastest. Another effective option is to use <b>ModF</b> with <b>ModR</b> and <b>PrymCrRNA1</b>. While <b>ModF</b> also works with <b>AltR</b> and <b>crRNA1</b>, or with <b>GalR</b> and <b>crRNA2</b>, these combinations yield lower intensity results. Therefore, the <b>ModF</b> and <b>GalR</b> primer pair is our top choice, as it demonstrates strong performance with both crRNAs and achieves the highest fluorescence intensity when combined with <b>crRNA1</b> among all the designs we tested.</p>
 +
 +
    <h2> Primer Concentration Optimization </h2>
 +
   
 +
<p>Since the ModF and GalR primers combined with PrymCrRNA1 proved to be our most effective combination, we decided to fine-tune the primer concentration to see if this adjustment could help us establish a correlation between fluorescence and the initial amount of DNA present in the sample. Our goal was to make the test quantifiable. </p>
 +
 +
<p>RPA reactions were set up, with different primer and input DNA concentrations, alongside a positive control (synDNA + SynCrRNA). For template dilutions, a PCR product coming from an algal culture was used. Dilutions of 200 nM, 2nM, 20 pM and 200 fM were tested, alongside the following primer concentrations: 960 nM, 480 nM, 240 nM and 120 nM. </p>
 +
 +
<h2> Limit of Detection</h2>
 +
<p> Based on the optimal primer concentration established in the previous assay, we conducted an additional test to determine the final limit of detection (LOD) for our assay.</p>
 +
 +
 +
 +
 +
    <h2>Detection of Prymnesium Types through Genomic Analysis</h2>
 +
    <p>Through genomic analysis, we have determined which types of <i>Prymnesium</i> our primer and crRNA combinations can detect.</p>
 +
   
 +
    <h3>Classification of Prymnesins</h3>
 +
    <p>Prymnesins are classified into three distinct types based on the structure of their carbon backbone [8]:</p>
 +
    <ul>
 +
        <li><b>A-type Prymnesins:</b> Have the largest carbon backbone with 91 carbon atoms. They are the most potent, exhibiting significant ichthyotoxic (fish-killing) properties.</li>
 +
        <li><b>B-type Prymnesins:</b> Feature a carbon backbone of 85 carbon atoms. They lack certain ring structures found in A-types and are generally less potent, though still toxic.</li>
 +
        <li><b>C-type Prymnesins:</b> Possess the shortest carbon backbone with 83 carbon atoms and show significant structural diversity. Their toxicity is variable and less well-characterized compared to A- and B-types.</li>
 +
    </ul>
 +
   
 +
    <h3 align="center">Table 1: Primer Combinations with ModF and Their Detection Capabilities</h3>
 +
    <table border="1" align="center" cellpadding="5" cellspacing="0">
 +
        <thead>
 +
            <tr>
 +
                <th align="center" style="font-size: smaller;">Primer Combination</th>
 +
                <th align="center" style="font-size: smaller;">Target Regions</th>
 +
                <th align="center" style="font-size: smaller;">Detected Types</th>
 +
            </tr>
 +
        </thead>
 +
        <tbody>
 +
            <tr>
 +
                <td align="center">ModF & ModR</td>
 +
                <td align="center">5.8S & ITS2</td>
 +
                <td align="center">Type B</td>
 +
            </tr>
 +
            <tr>
 +
                <td align="center">ModF & AltR</td>
 +
                <td align="center">5.8S & ITS2</td>
 +
                <td align="center">Type B</td>
 +
            </tr>
 +
            <tr>
 +
                <td align="center">ModF & GalR</td>
 +
                <td align="center">5.8S & ITS2</td>
 +
                <td align="center">All Types</td>
 +
            </tr>
 +
        </tbody>
 +
    </table>
 +
   
 +
    <h3 align="center">Table 2: crRNA Combinations and Their Detection Capabilities</h3>
 +
    <table border="1" align="center" cellpadding="5" cellspacing="0">
 +
        <thead>
 +
            <tr>
 +
                <th align="center" style="font-size: smaller;">crRNA Combination</th>
 +
                <th align="center" style="font-size: smaller;">Target Region</th>
 +
                <th align="center" style="font-size: smaller;">Detected Types</th>
 +
            </tr>
 +
        </thead>
 +
        <tbody>
 +
            <tr>
 +
                <td align="center">PrymCrRNA1</td>
 +
                <td align="center">ITS2</td>
 +
                <td align="center">Type A and B</td>
 +
            </tr>
 +
            <tr>
 +
                <td align="center">PrymCrRNA2</td>
 +
                <td align="center">ITS2</td>
 +
                <td align="center">Type A, B, and some C</td>
 +
            </tr>
 +
        </tbody>
 +
    </table>
 +
   
 +
    <h1>Sequence Source and Design</h1>
 +
    <p>This sequence was introduced by Luo et al. [6] and is intended for ITS sequencing of <i>Prymnesium parvum</i>. ModF is an unmodified version of the PR-RPA-4-F primer described in their publication. According to the authors, PR-RPA-4-F and its complementary PR-RPA-4-R primer were chosen as the best options for RPA reactions.</p>
 +
 +
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
 
<partinfo>BBa_K5087002 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K5087002 SequenceAndFeatures</partinfo>
 +
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 +
    <h2>Biosafety</h2>
 +
    <p>We used the Asimov's tool &mdash; Kernel &mdash; to check the sequence's safety with the <strong>Biosecurity Sequence Scanner</strong>. The results showed <strong>no flagged sequences</strong>, confirming that this part is safe to use.</p>
  
 +
    <h1>Resources</h1>
 +
    <ul>
 +
        <li>[1] White, T.J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications. Academic Press.</li>
 +
        <li>[2] Akhoundi, M., Downing, T., Votýpka, J., Kuhls, K., Lukeš, J., Cannet, A., Ravel, C., Marty, P., Delaunay, P., Kasbari, M., Granouillac, B., Gradoni, L., & Sereno, D. (2017). Leishmania infections: Molecular targets and diagnosis. <i>Molecular Aspects of Medicine, 57</i>, 1-29. https://doi.org/10.1016/j.mam.2016.11.012</li>
 +
        <li>[3] Galluzzi, L., Bertozzini, E., Penna, A., et al. (2008). Detection and quantification of <i>Prymnesium parvum</i> (Haptophyceae) by real-time PCR. <i>Letters in Applied Microbiology, 46</i>(2), 261-266. https://doi.org/10.1111/j.1472-765X.2007.02294.x</li>
 +
        <li>[4] Lobato, I. M., & O'Sullivan, C. K. (2018). Recombinase polymerase amplification: Basics, applications and recent advances. <i>Trends in Analytical Chemistry: TRAC, 98</i>, 19–35. https://doi.org/10.1016/j.trac.2017.10.015</li>
 +
        <li>[5] Ruichen Lv, Nianhong Lu and Junhu Wang et al. Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens: An Overview. <i>Zoonoses, 2</i>(1). DOI: 10.15212/ZOONOSES-2022-0002</li>
 +
        <li>[6] Luo N, Huang H, Jiang H. Establishment of methods for rapid detection of <i>Prymnesium parvum</i> by recombinase polymerase amplification combined with a lateral flow dipstick. <i>Frontiers in Marine Science, 9</i>. Available from: https://doi.org/10.3389/fmars.2022.1032847</li>
 +
        <li>[7] Kellner, Max J., Jeremy G. Koob, Jonathan S. Gootenberg, Omar O. Abudayyeh, and Feng Zhang. “SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases.” Nature Protocols 14, no. 10 (October 2019): 2986–3012. https://doi.org/10.1038/s41596-019-0210-2</li>
 +
        <li>[8] Binzer SB, Svenssen DK, Daugbjerg N, et al. A-, B- and C-type prymnesins are clade specific compounds and chemotaxonomic markers in Prymnesium parvum. Harmful Algae. 2019;81:10-17. doi:10.1016/j.hal.2018.11.010</li>
 +
    </ul>
  
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===Functional Parameters===
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<partinfo>BBa_K5087002 parameters</partinfo>
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Revision as of 11:34, 11 September 2024

ModF Part of the PrymDetect Toolkit

Introduction

ModF is a forward primer specifically designed to amplify a fragment of Prymnesium parvum genomic DNA in an RPA (Recombinase Polymerase Amplification) reaction by targeting the ITS2 region in the genome.

This primer contains a T7 promoter to enable in vitro transcription of the amplified product into RNA, preparing it for SHERLOCK detection with Cas13 proteins.

Biology and Usage

ITS Sequences

The ITS2 (Internal Transcribed Spacer 2) region is a non-coding segment of DNA found within the ribosomal RNA (rRNA) gene cluster. In the genome of Prymnesium parvum, the ITS2 region lies between the 5.8S and nuclear large rRNA genes [1].

The ITS regions, including ITS2, are commonly used for species identification because they tend to vary between species while being conserved within a species [2]. This variability makes the ITS2 region an effective target for designing species-specific primers, such as those used to identify Prymnesium parvum [3].

RPA Reaction

Recombinase Polymerase Amplification (RPA) is an isothermal nucleic acid amplification technique that operates at a temperature range of 37–42°C, distinguishing it from traditional PCR methods that require thermal cycling for denaturation and annealing of DNA [4].

RPA relies on three essential types of proteins: a recombinase, single-stranded DNA binding proteins (SSBs), and a strand-displacing DNA polymerase.

The process begins when the recombinase protein binds to a primer (about 30–35 nucleotides long) that matches the target DNA sequence. This complex then searches for homologous sequences in double-stranded DNA and initiates strand invasion. The SSBs stabilize the displaced strand to prevent primer dissociation, while the strand-displacing DNA polymerase extends the primer, resulting in exponential amplification of the target sequence [5].

RPA Mechanism
Figure 1. The RPA (Recombinase Polymerase Amplification) Mechanism

SHERLOCK Method

The SHERLOCK platform is a modern synthetic biology tool that utilizes the properties of the Cas13a protein, an enzyme from the Nobel Prize-winning CRISPR-Cas system. The Cas13a protein is guided with high specificity to the target sequence using crRNA.

The crRNA molecule is crucial for the assay's specificity. It consists of a direct repeat (DR) sequence and a spacer sequence that is complementary to the target. The crRNA molecule is designed to uniquely identify the organism by targeting the Internal Transcribed Spacer (ITS) sequence in its genome.

First, the Cas13a protein binds to the organism's genetic material, which was previously amplified using RPA and transcribed into RNA. Once bound, the Cas13a protein is activated and exhibits a “collateral” RNase activity, meaning it non-specifically cleaves nearby single-stranded RNA molecules [7].

This activity can be used in assays by including synthetic RNA probes tagged with a fluorescent reporter and a quencher in the reaction mixture. A fluorescent signal indicates that the reporters have been cleaved by Cas13, confirming the presence of the DNA target in the sample. The SHERLOCK method can also be used with Lateral Flow Assays (LFA).

SHERLOCK Method
Figure 2. The SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) Method Mechanism

Part Performance

This part is an integral component of the PrymDetect Toolkit, which is designed for detecting Prymnesium parvum in water samples using the SHERLOCK method. Our team evaluated this part alongside other components of the toolkit to determine the most reliable configurations and make its use easier for future iGEM teams.

Fluorescence Readout
Figure 3: Fluorescence readout results demonstrating the performance of various part combinations from our toolkit, with the ModF primer serving as the connecting component. For simplicity, the negative controls for both crRNAs are not shown, as they were all negative as anticipated.

Parts Compatible with ModF

The results show that ModF performs best when paired with the GalR primer and the PrymCrRNA1 design. This combination delivers the highest fluorescence readout intensity and provides results the fastest. Another effective option is to use ModF with ModR and PrymCrRNA1. While ModF also works with AltR and crRNA1, or with GalR and crRNA2, these combinations yield lower intensity results. Therefore, the ModF and GalR primer pair is our top choice, as it demonstrates strong performance with both crRNAs and achieves the highest fluorescence intensity when combined with crRNA1 among all the designs we tested.

Primer Concentration Optimization

Since the ModF and GalR primers combined with PrymCrRNA1 proved to be our most effective combination, we decided to fine-tune the primer concentration to see if this adjustment could help us establish a correlation between fluorescence and the initial amount of DNA present in the sample. Our goal was to make the test quantifiable.

RPA reactions were set up, with different primer and input DNA concentrations, alongside a positive control (synDNA + SynCrRNA). For template dilutions, a PCR product coming from an algal culture was used. Dilutions of 200 nM, 2nM, 20 pM and 200 fM were tested, alongside the following primer concentrations: 960 nM, 480 nM, 240 nM and 120 nM.

Limit of Detection

Based on the optimal primer concentration established in the previous assay, we conducted an additional test to determine the final limit of detection (LOD) for our assay.

Detection of Prymnesium Types through Genomic Analysis

Through genomic analysis, we have determined which types of Prymnesium our primer and crRNA combinations can detect.

Classification of Prymnesins

Prymnesins are classified into three distinct types based on the structure of their carbon backbone [8]:

  • A-type Prymnesins: Have the largest carbon backbone with 91 carbon atoms. They are the most potent, exhibiting significant ichthyotoxic (fish-killing) properties.
  • B-type Prymnesins: Feature a carbon backbone of 85 carbon atoms. They lack certain ring structures found in A-types and are generally less potent, though still toxic.
  • C-type Prymnesins: Possess the shortest carbon backbone with 83 carbon atoms and show significant structural diversity. Their toxicity is variable and less well-characterized compared to A- and B-types.

Table 1: Primer Combinations with ModF and Their Detection Capabilities

Primer Combination Target Regions Detected Types
ModF & ModR 5.8S & ITS2 Type B
ModF & AltR 5.8S & ITS2 Type B
ModF & GalR 5.8S & ITS2 All Types

Table 2: crRNA Combinations and Their Detection Capabilities

crRNA Combination Target Region Detected Types
PrymCrRNA1 ITS2 Type A and B
PrymCrRNA2 ITS2 Type A, B, and some C

Sequence Source and Design

This sequence was introduced by Luo et al. [6] and is intended for ITS sequencing of Prymnesium parvum. ModF is an unmodified version of the PR-RPA-4-F primer described in their publication. According to the authors, PR-RPA-4-F and its complementary PR-RPA-4-R primer were chosen as the best options for RPA reactions.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

Biosafety

We used the Asimov's tool — Kernel — to check the sequence's safety with the Biosecurity Sequence Scanner. The results showed no flagged sequences, confirming that this part is safe to use.

Resources

  • [1] White, T.J., Bruns, T., Lee, S., & Taylor, J. (1990). Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In PCR Protocols: A Guide to Methods and Applications. Academic Press.
  • [2] Akhoundi, M., Downing, T., Votýpka, J., Kuhls, K., Lukeš, J., Cannet, A., Ravel, C., Marty, P., Delaunay, P., Kasbari, M., Granouillac, B., Gradoni, L., & Sereno, D. (2017). Leishmania infections: Molecular targets and diagnosis. Molecular Aspects of Medicine, 57, 1-29. https://doi.org/10.1016/j.mam.2016.11.012
  • [3] Galluzzi, L., Bertozzini, E., Penna, A., et al. (2008). Detection and quantification of Prymnesium parvum (Haptophyceae) by real-time PCR. Letters in Applied Microbiology, 46(2), 261-266. https://doi.org/10.1111/j.1472-765X.2007.02294.x
  • [4] Lobato, I. M., & O'Sullivan, C. K. (2018). Recombinase polymerase amplification: Basics, applications and recent advances. Trends in Analytical Chemistry: TRAC, 98, 19–35. https://doi.org/10.1016/j.trac.2017.10.015
  • [5] Ruichen Lv, Nianhong Lu and Junhu Wang et al. Recombinase Polymerase Amplification for Rapid Detection of Zoonotic Pathogens: An Overview. Zoonoses, 2(1). DOI: 10.15212/ZOONOSES-2022-0002
  • [6] Luo N, Huang H, Jiang H. Establishment of methods for rapid detection of Prymnesium parvum by recombinase polymerase amplification combined with a lateral flow dipstick. Frontiers in Marine Science, 9. Available from: https://doi.org/10.3389/fmars.2022.1032847
  • [7] Kellner, Max J., Jeremy G. Koob, Jonathan S. Gootenberg, Omar O. Abudayyeh, and Feng Zhang. “SHERLOCK: Nucleic Acid Detection with CRISPR Nucleases.” Nature Protocols 14, no. 10 (October 2019): 2986–3012. https://doi.org/10.1038/s41596-019-0210-2
  • [8] Binzer SB, Svenssen DK, Daugbjerg N, et al. A-, B- and C-type prymnesins are clade specific compounds and chemotaxonomic markers in Prymnesium parvum. Harmful Algae. 2019;81:10-17. doi:10.1016/j.hal.2018.11.010