Difference between revisions of "Part:BBa K5088675"
 
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<partinfo>BBa_K5088675 short</partinfo>
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<partinfo>BBa_K5088675 short</partinfo>
 
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<html>
 
<html>
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==Background==
 
==Background==
 
<html>
 
<html>
−
the expression system used the GFP from the plant collection in the iGEM distribution kit. The expression was driven by the 35S CaMV promoter, TMV 5'UTR, and 35S 3'UTR. This system is widely used for constitutive overexpression of transgenes in various plant species, including model organisms and economically important crops (<a href="https://doi.org/10.1016/j.cpb.2020.100179" target=blank_>1</a>).
+
For initial testing of our measurement system we used the <a href="https://parts.igem.org/Part:BBa_J428076" target=blank_>GFP from the plant collection in the iGEM distribution kit</a>. The expression was driven by the 35S CaMV promoter, TMV 5'UTR, and 35S 3'UTR. This system is widely used for constitutive overexpression of transgenes in various plant species, including model organisms and economically important crops (<a href="https://doi.org/10.1016/j.cpb.2020.100179" target=blank_>1</a>).
−
 
+
</html>
  
  
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==Results==
 
==Results==
−
 
+
<html>
 +
<center>
 
<figure>
 
<figure>
 
         <a href="https://static.igem.wiki/teams/5088/wiki/images/engineering/pgfp-sbol.png" target="_blank">
 
         <a href="https://static.igem.wiki/teams/5088/wiki/images/engineering/pgfp-sbol.png" target="_blank">
−
             <img src="https://static.igem.wiki/teams/5088/wiki/images/engineering/pgfp-sbol.png" width="900px" max-height:400px>
+
             <img src="https://static.igem.wiki/teams/5088/wiki/images/engineering/pgfp-sbol.png" width=500px>
 
         </a>
 
         </a>
 
         <figcaption><p><b>Figure 1:</b> SBOL scheme of <a href="https://parts.igem.org/Part:BBa_K5088675" target=blank_>Tarakate - Test construct GFP [BBa_K5088675]</a>.</figcaption>
 
         <figcaption><p><b>Figure 1:</b> SBOL scheme of <a href="https://parts.igem.org/Part:BBa_K5088675" target=blank_>Tarakate - Test construct GFP [BBa_K5088675]</a>.</figcaption>
 
</figure>
 
</figure>
 +
</center>
 +
 +
 +
<center>
 +
<figure>
 +
        <a href="https://static.igem.wiki/teams/5088/registry/contribution/nb-pgfp.png" target="_blank">
 +
            <img src="https://static.igem.wiki/teams/5088/registry/contribution/nb-pgfp.png" width=900px>
 +
        </a>
 +
        <figcaption><p><b>Figure 2</b>: WT <i>N. benthamiana</i> plants treated with <i>A. rhizogenes</i> K599 and <i>A. tumfaciens</i> GV3101 P19 (negative control) under A) normal light conditions and B) under long-wave UV light (395nm). <i>N. benthamiana</i> infiltrated with <a href="https://parts.igem.org/Part:BBa_K5088675" target=blank_>Tarakate - Test construct GFP [BBa_K5088675]</a> under C) normal light conditions and D) under long-wave UV light (395nm).</figcaption>
 +
</figure>
 +
</center>
 +
 +
<center>
 +
<figure>
 +
        <a href="https://static.igem.wiki/teams/5088/wiki/images/engineering/nb-pgfp.png" target="_blank">
 +
            <img src="https://static.igem.wiki/teams/5088/wiki/images/engineering/nb-pgfp.png" width=900px>
 +
        </a>
 +
        <figcaption><p><b>Figure 3</b>: Successful expression of avGFP in N. benthamiana leaf disk assay using <a href="https://parts.igem.org/Part:BBa_K5088675" target=blank_>Tarakate - Test construct GFP [BBa_K5088675]</a>.</figcaption>
 +
</figure>
 +
</center>
 +
 +
 +
<center>
 +
<figure>
 +
        <a href="https://static.igem.wiki/teams/5088/wiki/images/engineering/protoplasts-gfp-pgfp.webp" target="_blank">
 +
            <img src="https://static.igem.wiki/teams/5088/wiki/images/engineering/protoplasts-gfp-pgfp.webp" width=900px>
 +
        </a>
 +
        <figcaption><p><b>Figure 4</b>: Successful expression of avGFP in protoplasts of Taraxacum officinale using <a href="https://parts.igem.org/Part:BBa_K5088675" target=blank_>Tarakate - Test construct GFP [BBa_K5088675]</a>.</figcaption>
 +
</figure>
 +
</center>
 +
 +
<center>
 +
<figure>
 +
        <a href="https://static.igem.wiki/teams/5088/wiki/images/engineering/cdb-gfp-root.png" target="_blank">
 +
            <img src="https://static.igem.wiki/teams/5088/wiki/images/engineering/cdb-gfp-root.png" width=900px>
 +
        </a>
 +
        <figcaption><p><b>Figure 5</b>: Successful expression of avGFP in roots of T. kok-saghyz using the CDB protocol and <a href="https://parts.igem.org/Part:BBa_K5088675" target=blank_>Tarakate - Test construct GFP [BBa_K5088675]</a>. A) Brightfield view of a transformed root of T. kok-saghyz B) The same root imaged under UV light.</figcaption>
 +
</figure>
 +
</center>
 +
</html>
 +
 +
 +
==The Dandelion Toolbox==
 +
<html>
 +
    <center>
 +
     
 +
<figure>
 +
<a href="https://static.igem.wiki/teams/5088/registry/illustrations/focusstacking-russiondandelion-white.jpg" target="_blank">
 +
    <img src="https://static.igem.wiki/teams/5088/registry/illustrations/focusstacking-russiondandelion-white.jpg" width="600px" max-height:400px>
 +
</a>
 +
</figure>
 +
</center> 
 +
 +
<p>
 +
    Our project aimed to advance the genetic engineering of dandelions by developing a robust set of constitutive regulatory parts. Using a transcriptomic approach, we identified 40 endogenous elements. To ensure precise and reliable testing, we constructed a ratiometric measurement system, enabling effective and quantitative characterization of these parts.
 +
</p>
 +
<p>
 +
    We employed three distinct plant transformation methods to test and validate the functionality of the regulatory elements. Through rigorous testing, we successfully characterized 23 out of the initial 40 elements, resulting in a comprehensive collection of standardized dandelion parts. This well-characterized suite of parts is designed to streamline future complex genetic engineering projects.
 +
</p>
 +
<p>
 +
    By providing these standardized tools, our project significantly lowers the barriers for researchers and iGEM teams, making Taraxacum kok-saghyz a more accessible and versatile chassis for plant synthetic biology. Ultimately, our work contributes to enhancing dandelion as a model organism and supporting sustainable natural rubber production.
 +
</p>
 +
 +
 +
</html>
 +
 +
 +
===Overview===
 +
<html>
 +
  <head>
 +
      <style>
 +
        #toolbox {
 +
        font-family: Arial, Helvetica, sans-serif;
 +
        border-collapse: collapse;
 +
        width: 100%;
 +
        }
 +
        #toolbox td, #customers th {
 +
        border: 1px solid #ddd;
 +
        padding: 8px;
 +
        }
 +
        #toolbox tr:nth-child(even){background-color: #f2f2f2;}
 +
        #toolbox tr:hover {background-color: #ddd;}
 +
        #toolbox th {
 +
        padding: 8px;
 +
        padding-top: 12px;
 +
        padding-bottom: 12px;
 +
        text-align: left;
 +
        background-color: #04AA6D;
 +
        color: white;
 +
        }
 +
      </style>
 +
  </head>
 +
  <body>
 +
      <table id="toolbox">
 +
        <tr>
 +
            <th>Part Identifier</th>
 +
            <th>Part Type</th>
 +
            <th>Nickname</th>
 +
            <th>Part Description</th>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088001">BBa_K5088001</a></td>
 +
            <td>Promoter + 5'UTR</td>
 +
            <td>P_RPL28</td>
 +
            <td>Large subunit ribosomal protein L28e - Promoter+5'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088006">BBa_K5088006</a></td>
 +
            <td>Promoter + 5'UTR</td>
 +
            <td>P_FKBP4_5</td>
 +
            <td>FK506-binding protein 4/5 - Promoter+5'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088007">BBa_K5088007</a></td>
 +
            <td>Promoter + 5'UTR</td>
 +
            <td>P_CLTC</td>
 +
            <td>Clathrin - Promoter+5'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088008">BBa_K5088008</a></td>
 +
            <td>Promoter + 5'UTR</td>
 +
            <td>P_RPL31</td>
 +
            <td>Large subunit ribosomal protein L31e - Promoter+5'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088012">BBa_K5088012</a></td>
 +
            <td>Promoter + 5'UTR</td>
 +
            <td>P_Tubulin</td>
 +
            <td>Tubulin - Promoter+5'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088013">BBa_K5088013</a></td>
 +
            <td>Promoter + 5'UTR</td>
 +
            <td>P_EIF5A</td>
 +
            <td>Translation initiation factor 5A - Promoter+5'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088102">BBa_K5088102</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_PTI1</td>
 +
            <td>Protein tyrosine kinase - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088103">BBa_K5088103</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_RPL28</td>
 +
            <td>Large subunit ribosomal protein L28e - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088104">BBa_K5088104</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_EPS15</td>
 +
            <td>Epidermal growth factor receptor substrate 15 - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088105">BBa_K5088105</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_GSK3B</td>
 +
            <td>Glycogen synthase kinase 3 - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088106">BBa_K5088106</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_MGRN1</td>
 +
            <td>E3 ubiquitin-protein ligase - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088107">BBa_K5088107</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_RPL35A</td>
 +
            <td>Large subunit ribosomal protein L35Ae - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088108">BBa_K5088108</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_betB</td>
 +
            <td>Betaine-aldehyde dehydrogenase - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088109">BBa_K5088109</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_pgm</td>
 +
            <td>Phosphoglucomutase - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088110">BBa_K5088110</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_ATP-synt</td>
 +
            <td>ATPase subunit gamma - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088111">BBa_K5088111</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_EIF3B</td>
 +
            <td>Translation initiation factor 3 subunit B - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088112">BBa_K5088112</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_RPL31</td>
 +
            <td>Large subunit ribosomal protein L31e - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088113">BBa_K5088113</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_TM9SF2_4</td>
 +
            <td>Transmembrane 9 superfamily member 2/4 - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088114">BBa_K5088114</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_CUL1</td>
 +
            <td>Cullin - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088115">BBa_K5088115</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_PSMB6</td>
 +
            <td>20S proteasome subunit beta 1 - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088116">BBa_K5088116</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_RPSA</td>
 +
            <td>Small subunit ribosomal protein SAe - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088117">BBa_K5088117</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_VPS4</td>
 +
            <td>Vacuolar protein-sorting-associated protein 4 - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
        <tr>
 +
            <td><a href="https://parts.igem.org/Part:BBa_K5088118">BBa_K5088118</a></td>
 +
            <td>3'UTR</td>
 +
            <td>T_EIF2S3</td>
 +
            <td>Translation initiation factor 2 subunit 3 - 3'UTR from <i>T. kok-saghyz</i></td>
 +
        </tr>
 +
      </table>
 +
<caption><b>Table 1:</b> List of functioning <i>T. kok-saghyz</i> endogenous regulatory elements we've characterized in our project</caption>
 +
  </body>
 +
</html>
 +
 +
===Dandelion Handbook===
 +
<html>
 +
  <p>By creating a suite of genetic tools and transformation methods, and sharing them through our Dandelion Handbook, we believe that dandelions can serve as an excellent chassis for numerous applications. We aim to inspire future iGEM teams to harness the unique properties of dandelions for a variety of promising projects.</p>
 +
  <p>Dandelions have demonstrated their versatility, being used as a coffee alternative and in various food applications such as salads, wine, and honey. Additionally, their ability to naturally hyperaccumulate environmental pollutants, including heavy metals, highlights their potential for bioremediation applications.</p>
 +
  <p>By equipping future iGEM teams with these resources, we aspire to unlock the full potential of dandelions, paving the way for sustainable and diverse synthetic biology applications.</p>
 +
<p><a href="https://2024.igem.wiki/marburg/handbook">Click here to look at our Dandelion Handbook</a></p>.
 +
</html>

Latest revision as of 22:02, 1 October 2024

Tarakate - Test construct GFP


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 230
    Illegal XhoI site found at 1257
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Background

For initial testing of our measurement system we used the GFP from the plant collection in the iGEM distribution kit. The expression was driven by the 35S CaMV promoter, TMV 5'UTR, and 35S 3'UTR. This system is widely used for constitutive overexpression of transgenes in various plant species, including model organisms and economically important crops (1).



Results

Figure 1: SBOL scheme of Tarakate - Test construct GFP [BBa_K5088675].

Figure 2: WT N. benthamiana plants treated with A. rhizogenes K599 and A. tumfaciens GV3101 P19 (negative control) under A) normal light conditions and B) under long-wave UV light (395nm). N. benthamiana infiltrated with Tarakate - Test construct GFP [BBa_K5088675] under C) normal light conditions and D) under long-wave UV light (395nm).

Figure 3: Successful expression of avGFP in N. benthamiana leaf disk assay using Tarakate - Test construct GFP [BBa_K5088675].

Figure 4: Successful expression of avGFP in protoplasts of Taraxacum officinale using Tarakate - Test construct GFP [BBa_K5088675].

Figure 5: Successful expression of avGFP in roots of T. kok-saghyz using the CDB protocol and Tarakate - Test construct GFP [BBa_K5088675]. A) Brightfield view of a transformed root of T. kok-saghyz B) The same root imaged under UV light.


The Dandelion Toolbox

Our project aimed to advance the genetic engineering of dandelions by developing a robust set of constitutive regulatory parts. Using a transcriptomic approach, we identified 40 endogenous elements. To ensure precise and reliable testing, we constructed a ratiometric measurement system, enabling effective and quantitative characterization of these parts.

We employed three distinct plant transformation methods to test and validate the functionality of the regulatory elements. Through rigorous testing, we successfully characterized 23 out of the initial 40 elements, resulting in a comprehensive collection of standardized dandelion parts. This well-characterized suite of parts is designed to streamline future complex genetic engineering projects.

By providing these standardized tools, our project significantly lowers the barriers for researchers and iGEM teams, making Taraxacum kok-saghyz a more accessible and versatile chassis for plant synthetic biology. Ultimately, our work contributes to enhancing dandelion as a model organism and supporting sustainable natural rubber production.


Overview

Part Identifier Part Type Nickname Part Description
BBa_K5088001 Promoter + 5'UTR P_RPL28 Large subunit ribosomal protein L28e - Promoter+5'UTR from T. kok-saghyz
BBa_K5088006 Promoter + 5'UTR P_FKBP4_5 FK506-binding protein 4/5 - Promoter+5'UTR from T. kok-saghyz
BBa_K5088007 Promoter + 5'UTR P_CLTC Clathrin - Promoter+5'UTR from T. kok-saghyz
BBa_K5088008 Promoter + 5'UTR P_RPL31 Large subunit ribosomal protein L31e - Promoter+5'UTR from T. kok-saghyz
BBa_K5088012 Promoter + 5'UTR P_Tubulin Tubulin - Promoter+5'UTR from T. kok-saghyz
BBa_K5088013 Promoter + 5'UTR P_EIF5A Translation initiation factor 5A - Promoter+5'UTR from T. kok-saghyz
BBa_K5088102 3'UTR T_PTI1 Protein tyrosine kinase - 3'UTR from T. kok-saghyz
BBa_K5088103 3'UTR T_RPL28 Large subunit ribosomal protein L28e - 3'UTR from T. kok-saghyz
BBa_K5088104 3'UTR T_EPS15 Epidermal growth factor receptor substrate 15 - 3'UTR from T. kok-saghyz
BBa_K5088105 3'UTR T_GSK3B Glycogen synthase kinase 3 - 3'UTR from T. kok-saghyz
BBa_K5088106 3'UTR T_MGRN1 E3 ubiquitin-protein ligase - 3'UTR from T. kok-saghyz
BBa_K5088107 3'UTR T_RPL35A Large subunit ribosomal protein L35Ae - 3'UTR from T. kok-saghyz
BBa_K5088108 3'UTR T_betB Betaine-aldehyde dehydrogenase - 3'UTR from T. kok-saghyz
BBa_K5088109 3'UTR T_pgm Phosphoglucomutase - 3'UTR from T. kok-saghyz
BBa_K5088110 3'UTR T_ATP-synt ATPase subunit gamma - 3'UTR from T. kok-saghyz
BBa_K5088111 3'UTR T_EIF3B Translation initiation factor 3 subunit B - 3'UTR from T. kok-saghyz
BBa_K5088112 3'UTR T_RPL31 Large subunit ribosomal protein L31e - 3'UTR from T. kok-saghyz
BBa_K5088113 3'UTR T_TM9SF2_4 Transmembrane 9 superfamily member 2/4 - 3'UTR from T. kok-saghyz
BBa_K5088114 3'UTR T_CUL1 Cullin - 3'UTR from T. kok-saghyz
BBa_K5088115 3'UTR T_PSMB6 20S proteasome subunit beta 1 - 3'UTR from T. kok-saghyz
BBa_K5088116 3'UTR T_RPSA Small subunit ribosomal protein SAe - 3'UTR from T. kok-saghyz
BBa_K5088117 3'UTR T_VPS4 Vacuolar protein-sorting-associated protein 4 - 3'UTR from T. kok-saghyz
BBa_K5088118 3'UTR T_EIF2S3 Translation initiation factor 2 subunit 3 - 3'UTR from T. kok-saghyz
Table 1: List of functioning T. kok-saghyz endogenous regulatory elements we've characterized in our project

Dandelion Handbook

By creating a suite of genetic tools and transformation methods, and sharing them through our Dandelion Handbook, we believe that dandelions can serve as an excellent chassis for numerous applications. We aim to inspire future iGEM teams to harness the unique properties of dandelions for a variety of promising projects.

Dandelions have demonstrated their versatility, being used as a coffee alternative and in various food applications such as salads, wine, and honey. Additionally, their ability to naturally hyperaccumulate environmental pollutants, including heavy metals, highlights their potential for bioremediation applications.

By equipping future iGEM teams with these resources, we aspire to unlock the full potential of dandelions, paving the way for sustainable and diverse synthetic biology applications.

Click here to look at our Dandelion Handbook

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