Difference between revisions of "Part:BBa K1965033"

 
 
Line 3: Line 3:
 
<partinfo>BBa_K1965033 short</partinfo>
 
<partinfo>BBa_K1965033 short</partinfo>
  
−
description
+
<html lang="en">
 +
<head>
 +
    <meta charset="UTF-8">
 +
    <title></title>
 +
    <style>
 +
        .ui.medium.image {
 +
            width: 100%;
 +
        }
 +
    </style>
 +
    <script type="text/javascript">
 +
        $(this).find('ref').each(function () {
 +
            var label = $(this).text();
 +
            $(this).click(function (event) {
 +
                event.preventDefault();
 +
            });
 +
            var number = figures[1][label];
 +
            $(this).html("<a href=''>Figure " + figures[1][label] + "</a>");
 +
            $(this).on('click', function () {
 +
                window.scroll(0, $(figures[0][number]).offset().top + 100);
 +
            })
 +
        });
 +
        function enumerateKeys(object) {
 +
            var numberPairs = {};
 +
            var objectPairs = {};
 +
            var number = 0;
 +
 
 +
            var matches = document.body.querySelectorAll(object + '[data-ref]');
 +
            while (number < matches.length) {
 +
                var refName = matches[number].dataset.ref;
 +
                if (refName in numberPairs) {
 +
                    console.log(refName, "This label has already been assigned.");
 +
                } else {
 +
                    numberPairs[number + 1] = matches[number];
 +
                    objectPairs[refName] = number + 1;
 +
                }
 +
                number++;
 +
            }
 +
            return [numberPairs, objectPairs, number];
 +
        }
 +
 
 +
        function enumerate() {
 +
            var figures = enumerateKeys('figure');
 +
            var maximum = figures[2];
 +
 
 +
            for (var number = 1; number <= maximum; number++) {
 +
                if (number in figures[0]) {
 +
                    $(figures[0][number]).children('figcaption').prepend("Figure " + number + ": ")
 +
                }
 +
            }
 +
 
 +
            $(this).find('ref').each(function () {
 +
                var label = $(this).text();
 +
                $(this).click(function (event) {
 +
                    event.preventDefault();
 +
                });
 +
                var number = figures[1][label];
 +
                $(this).html("<a href=''>Figure " + figures[1][label] + "</a>");
 +
                $(this).on('click', function () {
 +
                    window.scroll(0, $(figures[0][number]).offset().top - 110);
 +
                })
 +
            });
 +
        }
 +
        $(document).ready(enumerate);
 +
    </script>
 +
</head>
 +
<body>
 +
<h3>Introduction </h3>
 +
<p>The split protein system based on inducible dimerization is an attractive method to regulate protease activity. Wehr et al. described a split tobacco etch virus protease (TEVp) expressed as two functionally inactive fragments; the N-terminal (1 – 118 aa) and C-terminal (119 – 242 aa) protease fragments (referred to as cTEVp and nTEVp) <sup>[1]</sup> .</p>
 +
<p>Soybean mosaic virus protease (SbMVp) is a 27kDa protease from soybean mosaic virus (SbMV). SbMVp is also known as nuclear inclusion a protein (NIa) and is one of the three viral proteases responsible for the processing of the viral polyprotein to at least ten functional segments <sup>[2,3]</sup>. SbMVp has been recently studied by Seo et al. <sup>[3]</sup>  as a tool for protein-protein interaction studies in the soybean. We converted SbMVp to split protease by splitting it at positions corresponding to the position of the previously described split TEV protease. </p>
 +
<p>FRB is a protein that binds the small molecule rapamycin with high affinity. In combination with the FK-506 binding protein (FKBP) it is widely used for induced dimerization of proteins. Proteins of interest can be fused to FKBP or FRB and then conditionally dimerized by the addition of rapamycin <sup>[4]</sup> (<a href="http://2016.igem.org/Team:Slovenia/Protease_signaling/Split_proteases">CID</a>). </p>
 +
<p>SbMVp has well defined seven amino acid recognition motif SbMVs which is dermined by amino acid sequence ESVSLQ-S. For a detailed description of SbMVp click <a href="https://parts.igem.org/Part:BBa_K1965040">BBa_K1965040</a>. </p>
 +
 
 +
<h3>Characterization</h3>
 +
<p>This part consist of the N-terminus of soybean mosaic virus protease (SbMVp) fused to FKBP-rapamycin binding (FRB) domain and works in combination with the part FKBP:cSbMVp (<a href="https://parts.igem.org/Part:BBa_K1965032">BBa_K1965032</a>). </p>
 +
<div align = "left">
 +
 
 +
    <figure data-ref="1">
 +
 
 +
        <img class="ui medium image" src=" https://static.igem.org/mediawiki/2016/4/41/T--Slovenia--BBa_K1965031%282%29.PNG" >
 +
<figcaption><b>Activitiy of split SbMVs based on rapamycin inducible system.</b><br/>HEK293T cells were trasnfected with 100 ng of the cycLuc_SbMVs reporter and 70 ng of each split SbMV fragment. The whole SbMVp (70 ng) was used as positive control. An increase in luciferase activity was detected in cells induced with rapamycin.</figcaption>
 +
    </figure>
 +
</div>
 +
 
 +
<p>We tested rapamycin inducible split SbMVp system by measuring activity with the cycLuc reporter (link). Increasing luciferase activity was detected, correlating with the amount of the transfected protease fragments in stimulated cells <ref>1</ref>. Luciferase in unstimulated cells remained inactive even at the highest amount of transfected protease fragments, proving low leakage and high inducibility of the split protease system in response to rapamycin.</p>
 +
 
 +
<h3>References:</h3>
 +
<sup>[1]</sup>Wehr, M. C. et al. Monitoring regulated protein-protein interactions using split TEV. Nat. Methods 3, 985–93 (2006).<br>
 +
<sup>[2]</sup>Adams, M. J., Antoniw, J. F. & Beaudoin, F. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Mol. Plant Pathol. 6, 471–87 (2005). <br>
 +
<sup>[3]</sup>Seo, J.-K. et al. Engineering of soybean mosaic virus as a versatile tool for studying protein–protein interactions in soybean. Sci. Rep. 6, 22436 (2016). <br>
 +
<sup>[4]</sup>Banaszynski, L. A., Liu, C. W. & Wandless, T. J. Characterization of the FKBP‚Rapamycin‚FRB Ternary Complex. doi:10.1021/ja043277y<br>
 +
 
 +
</body>
 +
</html>
 +
 
 +
 
 +
 
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 17:27, 18 October 2016


Myc:FRB:nSbMVp

Introduction

The split protein system based on inducible dimerization is an attractive method to regulate protease activity. Wehr et al. described a split tobacco etch virus protease (TEVp) expressed as two functionally inactive fragments; the N-terminal (1 – 118 aa) and C-terminal (119 – 242 aa) protease fragments (referred to as cTEVp and nTEVp) [1] .

Soybean mosaic virus protease (SbMVp) is a 27kDa protease from soybean mosaic virus (SbMV). SbMVp is also known as nuclear inclusion a protein (NIa) and is one of the three viral proteases responsible for the processing of the viral polyprotein to at least ten functional segments [2,3]. SbMVp has been recently studied by Seo et al. [3] as a tool for protein-protein interaction studies in the soybean. We converted SbMVp to split protease by splitting it at positions corresponding to the position of the previously described split TEV protease.

FRB is a protein that binds the small molecule rapamycin with high affinity. In combination with the FK-506 binding protein (FKBP) it is widely used for induced dimerization of proteins. Proteins of interest can be fused to FKBP or FRB and then conditionally dimerized by the addition of rapamycin [4] (CID).

SbMVp has well defined seven amino acid recognition motif SbMVs which is dermined by amino acid sequence ESVSLQ-S. For a detailed description of SbMVp click BBa_K1965040.

Characterization

This part consist of the N-terminus of soybean mosaic virus protease (SbMVp) fused to FKBP-rapamycin binding (FRB) domain and works in combination with the part FKBP:cSbMVp (BBa_K1965032).

Activitiy of split SbMVs based on rapamycin inducible system.
HEK293T cells were trasnfected with 100 ng of the cycLuc_SbMVs reporter and 70 ng of each split SbMV fragment. The whole SbMVp (70 ng) was used as positive control. An increase in luciferase activity was detected in cells induced with rapamycin.

We tested rapamycin inducible split SbMVp system by measuring activity with the cycLuc reporter (link). Increasing luciferase activity was detected, correlating with the amount of the transfected protease fragments in stimulated cells 1. Luciferase in unstimulated cells remained inactive even at the highest amount of transfected protease fragments, proving low leakage and high inducibility of the split protease system in response to rapamycin.

References:

[1]Wehr, M. C. et al. Monitoring regulated protein-protein interactions using split TEV. Nat. Methods 3, 985–93 (2006).
[2]Adams, M. J., Antoniw, J. F. & Beaudoin, F. Overview and analysis of the polyprotein cleavage sites in the family Potyviridae. Mol. Plant Pathol. 6, 471–87 (2005).
[3]Seo, J.-K. et al. Engineering of soybean mosaic virus as a versatile tool for studying protein–protein interactions in soybean. Sci. Rep. 6, 22436 (2016).
[4]Banaszynski, L. A., Liu, C. W. & Wandless, T. J. Characterization of the FKBP‚Rapamycin‚FRB Ternary Complex. doi:10.1021/ja043277y



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 313
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 412