Difference between revisions of "Team:Edinburgh UG/Parts"

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             <h2 class="header-subsection"> Basic parts </h2>
 
             <h2 class="header-subsection"> Basic parts </h2>
−
             <p> Our basic parts fall into three categories: Recombinase generators, recombinase target sites, and orthogonal target sites for Cre. We selected the recombinases Dre [1], Vika [2], VCre, and SCre [3] to form the basis of our toolkit. These recombinases were shown to not cross-react with the Cre/LoxP system [1][2][3][5], and, in the case of VCre and SCre [3], were claimed to not cross-react with one another. As part of our toolkit, we also included the associated target sites for these recombinase generators. For a snapshot of how we verified the function of these parts, scroll down to the “Demonstrate” section of this page. For a more detailed view, click on the parts registry link, which will take you to the detailed analysis of each individual part. We also included in the toolkit orthogonal target sites for Cre recombinase. Cre/LoxP is a very popular recombinase system that is frequently used in biotechnology [4]. In order to expand the potential of this system, we have developed multiple mutant Lox target sites, which have previously been identified as sites that will not react with the classic LoxP site [4]. Adding these mutant, orthogonal lox sites to the toolkits means we provide a framework whereby one Cre protein could catalyse up to ten distinct recombination events within one cell. Once again, check out the full registry pages for each part to learn more and download their individual sequences. To simplify our experimental workflows, we have also developed an inducible Cre recombinase generator, also reported below.  
+
             <p> Our basic parts fall into three categories: Recombinase generators, recombinase target sites, and orthogonal
 +
                target sites for Cre. We selected the recombinases Dre [1], Vika [2], VCre, and SCre [3] to form the basis
 +
                of our toolkit. These recombinases were shown to not cross-react with the Cre/LoxP system [1][2][3][5], and,
 +
                in the case of VCre and SCre [3], were claimed to not cross-react with one another. As part of our toolkit,
 +
                we also included the associated target sites for these recombinase generators. For a snapshot of how we verified
 +
                the function of these parts, scroll down to the “Demonstrate” section of this page. For a more detailed view,
 +
                click on the parts registry link, which will take you to the detailed analysis of each individual part. We
 +
                also included in the toolkit orthogonal target sites for Cre recombinase. Cre/LoxP is a very popular recombinase
 +
                system that is frequently used in biotechnology [4]. In order to expand the potential of this system, we
 +
                have developed multiple mutant Lox target sites, which have previously been identified as sites that will
 +
                not react with the classic LoxP site [4]. Adding these mutant, orthogonal lox sites to the toolkits means
 +
                we provide a framework whereby one Cre protein could catalyse up to ten distinct recombination events within
 +
                one cell. Once again, check out the full registry pages for each part to learn more and download their individual
 +
                sequences. To simplify our experimental workflows, we have also developed an inducible Cre recombinase generator,
 +
                also reported below.
 
             </p>
 
             </p>
  
Line 41: Line 55:
 
                     <td><a href="http://parts.igem.org/Part:BBa_K2406081"> http://parts.igem.org/Part:BBa_K2406081 </a> </td>
 
                     <td><a href="http://parts.igem.org/Part:BBa_K2406081"> http://parts.igem.org/Part:BBa_K2406081 </a> </td>
 
                 </tr>
 
                 </tr>
−
             </table>
+
                <tr>
 +
                    <td>T7-LacO-Vika Basic</td>
 +
                    <td>Recombinase generator</td>
 +
                    <td>Bba_K2406082</td>
 +
                    <td>Shown to excise terminator inducibly</td>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_K2406082"> http://parts.igem.org/Part:BBa_K2406082 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>T7-LacO-Vcre Basic</td>
 +
                    <td>Recombinase generator</td>
 +
                    <td>Bba_K2406083</td>
 +
                    <td>Shown to excise terminator inducibly</td>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_K2406083"> http://parts.igem.org/Part:BBa_K2406083 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>T7-LacO-Scre Basic</td>
 +
                    <td>Recombinase generator</td>
 +
                    <td>Bba_K2406084</td>
 +
                    <td>Shown to excise terminator inducibly</td>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_K2406084"> http://parts.igem.org/Part:BBa_K2406084 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>T7-LacO-Dre Basic</td>
 +
                    <td>Recombinase generator</td>
 +
                    <td>Bba_K2406081</td>
 +
                    <td>Shown to excise terminator inducibly</td>
 +
                    <td><a href="http://parts.igem.org/Part:BBa_K2406081"> http://parts.igem.org/Part:BBa_K2406081 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>Rox</td>
 +
                    <td>Recombinase target site</td>
 +
                    <td>BBa_K2406000</td>
 +
                    <td>Excise terminator, orthogonal test</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406000"> http://parts.igem.org/Part:Bba_K2406000 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>Vox</td>
 +
                    <td>Recombinase target site</td>
 +
                    <td>BBa_K2406001</td>
 +
                    <td>Excise terminator, orthogonal test</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406001"> http://parts.igem.org/Part:Bba_K2406001 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>Vlox</td>
 +
                    <td>Recombinase target site</td>
 +
                    <td>BBa_K2406002</td>
 +
                    <td>Excise terminator, orthogonal test</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406002"> http://parts.igem.org/Part:Bba_K2406002 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>Slox</td>
 +
                    <td>Recombinase target site</td>
 +
                    <td>BBa_K2406003</td>
 +
                    <td>Excise terminator, orthogonal test</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406003"> http://parts.igem.org/Part:Bba_K2406003 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>Lox511</td>
 +
                    <td>Orthogonal Lox site</td>
 +
                    <td>Bba_K2406008</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406008"> http://parts.igem.org/Part:Bba_K2406008 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>Lox2272</td>
 +
                    <td>Orthogonal Lox site</td>
 +
                    <td>Bba_K2406009</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406009"> http://parts.igem.org/Part:Bba_K2406009 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>Lox5171</td>
 +
                    <td>Orthogonal Lox site</td>
 +
                    <td>Bba_K2406010</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406010"> http://parts.igem.org/Part:Bba_K2406010 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>LoxN</td>
 +
                    <td>Orthogonal Lox site</td>
 +
                    <td>Bba_K2406011</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406011"> http://parts.igem.org/Part:Bba_K2406011 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>M2</td>
 +
                    <td>Orthogonal Lox site</td>
 +
                    <td>Bba_K2406012</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406012"> http://parts.igem.org/Part:Bba_K2406012 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>M3</td>
 +
                    <td>Orthogonal Lox site</td>
 +
                    <td>Bba_K2406013</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406013"> http://parts.igem.org/Part:Bba_K2406013 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>M7</td>
 +
                    <td>Orthogonal Lox site</td>
 +
                    <td>Bba_K2406014</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406014"> http://parts.igem.org/Part:Bba_K2406014 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>M11</td>
 +
                    <td>Orthogonal Lox site</td>
 +
                    <td>Bba_K2406015</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406015"> http://parts.igem.org/Part:Bba_K2406015 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>Nuoya</td>
 +
                    <td>Orthogonal Lox site</td>
 +
                    <td>Bba_K2406016</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406016"> http://parts.igem.org/Part:Bba_K2406016 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>Zsoka</td>
 +
                    <td>Orthogonal Lox site</td>
 +
                    <td>Bba_K2406017</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406017"> http://parts.igem.org/Part:Bba_K2406017 </a> </td>
 +
                </tr>
 +
                <tr>
 +
                    <td>T7-LacO-Cre</td>
 +
                    <td>Recombinase generator</td>
 +
                    <td>Bba_K2406080</td>
 +
                    <td>Sequencing, functional assay</td>
 +
                    <td><a href="http://parts.igem.org/Part:Bba_K2406080"> http://parts.igem.org/Part:Bba_K2406080 </a> </td>
 +
                </tr>
 +
             </table><br>
 +
 
 +
 
 +
            <h2 class="header-subsection"> Composite parts </h2>
 +
            <p> Our generators use our novel T7-LacO promoter composite. This allows for inducible expression of our parts in E. coli strains expressing T7 Polymerase. As is elaborated on the improve section of this page, this constitutes a valuable part improvement. Finally, we assembled measurement constructs. These consisted of two target sites flanking a promoter, with this part inserted in between the promoter and RBS of the BBa_J04450 part. As explained in the diagram below, these constructs could test if two target sites would recombine when a particular recombinase was present in the cell. This allowed us to demonstrate our recombinase target sites, recombinase coding sequences, and novel promoter functioned as anticipated. Furthermore, they allowed for us to demonstrate which target sites for different recombinases would not recombine with one another. It should be noted we did observe some unexpected cross-reactivity. In a sense, this is a positive, as it verifies the validity of our measurement construct for testing if two recombinase sites are orthogonal. Indeed, it appears our measurement constructs are more sensitive than those reported in the literature, as cross reactivity for SCre and VCre had not previously been observed [3].
 +
            </p>
 +
 
 
             <figure style="text-align: center;">
 
             <figure style="text-align: center;">
−
                 <img src="https://static.igem.org/mediawiki/2017/0/0d/T--Edinburgh_UG--Interlab3.jpg">
+
                 <img src="https://static.igem.org/mediawiki/2017/f/f6/T--Edinburgh_UG--Parts1.jpg">
 +
                <figcaption> Fig. 1: Basic schematic demonstrating principle of our recombinase measurement constructs. Parallel lines (or capacitor for engineers) represents transcription terminator downstream of a promoter. Triangles of identical colours represent target sites that can recombine with one another. Different coloured triangles are incompatible target sites. Note that RFP reporter gives off quantitative output, so recombination efficiency between two sites can also be quantified. </figcaption>
 
             </figure>
 
             </figure>
  

Revision as of 23:02, 30 October 2017




Parts

Introduction

The main goal of our project in the short-term was to develop and test recombinase parts in E. coli. To the best of our knowledge, these parts have not been extensively tested for activity and orthogonality in bacterial cells. The parts listed below have all been functionally verified, and can be used to construct the systems described on our design page [link]. We report verified function in four tyrosine recombinases, none of which have been correctly documented by any iGEM team before. We also report target sites for each recombinase, none of which have been properly documented or entered into the registry before. We have also developed a collection of orthogonal Cre-recognised Lox sites. Finally, we have developed measurement constructs for each recombinase. We used these constructs to measure and confirm the activity of our basic parts.

Basic parts

Our basic parts fall into three categories: Recombinase generators, recombinase target sites, and orthogonal target sites for Cre. We selected the recombinases Dre [1], Vika [2], VCre, and SCre [3] to form the basis of our toolkit. These recombinases were shown to not cross-react with the Cre/LoxP system [1][2][3][5], and, in the case of VCre and SCre [3], were claimed to not cross-react with one another. As part of our toolkit, we also included the associated target sites for these recombinase generators. For a snapshot of how we verified the function of these parts, scroll down to the “Demonstrate” section of this page. For a more detailed view, click on the parts registry link, which will take you to the detailed analysis of each individual part. We also included in the toolkit orthogonal target sites for Cre recombinase. Cre/LoxP is a very popular recombinase system that is frequently used in biotechnology [4]. In order to expand the potential of this system, we have developed multiple mutant Lox target sites, which have previously been identified as sites that will not react with the classic LoxP site [4]. Adding these mutant, orthogonal lox sites to the toolkits means we provide a framework whereby one Cre protein could catalyse up to ten distinct recombination events within one cell. Once again, check out the full registry pages for each part to learn more and download their individual sequences. To simplify our experimental workflows, we have also developed an inducible Cre recombinase generator, also reported below.

T7-LacO-Dre Basic Recombinase generator Bba_K2406081 Shown to excise terminator inducibly http://parts.igem.org/Part:BBa_K2406081
T7-LacO-Vika Basic Recombinase generator Bba_K2406082 Shown to excise terminator inducibly http://parts.igem.org/Part:BBa_K2406082
T7-LacO-Vcre Basic Recombinase generator Bba_K2406083 Shown to excise terminator inducibly http://parts.igem.org/Part:BBa_K2406083
T7-LacO-Scre Basic Recombinase generator Bba_K2406084 Shown to excise terminator inducibly http://parts.igem.org/Part:BBa_K2406084
T7-LacO-Dre Basic Recombinase generator Bba_K2406081 Shown to excise terminator inducibly http://parts.igem.org/Part:BBa_K2406081
Rox Recombinase target site BBa_K2406000 Excise terminator, orthogonal test http://parts.igem.org/Part:Bba_K2406000
Vox Recombinase target site BBa_K2406001 Excise terminator, orthogonal test http://parts.igem.org/Part:Bba_K2406001
Vlox Recombinase target site BBa_K2406002 Excise terminator, orthogonal test http://parts.igem.org/Part:Bba_K2406002
Slox Recombinase target site BBa_K2406003 Excise terminator, orthogonal test http://parts.igem.org/Part:Bba_K2406003
Lox511 Orthogonal Lox site Bba_K2406008 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406008
Lox2272 Orthogonal Lox site Bba_K2406009 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406009
Lox5171 Orthogonal Lox site Bba_K2406010 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406010
LoxN Orthogonal Lox site Bba_K2406011 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406011
M2 Orthogonal Lox site Bba_K2406012 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406012
M3 Orthogonal Lox site Bba_K2406013 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406013
M7 Orthogonal Lox site Bba_K2406014 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406014
M11 Orthogonal Lox site Bba_K2406015 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406015
Nuoya Orthogonal Lox site Bba_K2406016 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406016
Zsoka Orthogonal Lox site Bba_K2406017 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406017
T7-LacO-Cre Recombinase generator Bba_K2406080 Sequencing, functional assay http://parts.igem.org/Part:Bba_K2406080

Composite parts

Our generators use our novel T7-LacO promoter composite. This allows for inducible expression of our parts in E. coli strains expressing T7 Polymerase. As is elaborated on the improve section of this page, this constitutes a valuable part improvement. Finally, we assembled measurement constructs. These consisted of two target sites flanking a promoter, with this part inserted in between the promoter and RBS of the BBa_J04450 part. As explained in the diagram below, these constructs could test if two target sites would recombine when a particular recombinase was present in the cell. This allowed us to demonstrate our recombinase target sites, recombinase coding sequences, and novel promoter functioned as anticipated. Furthermore, they allowed for us to demonstrate which target sites for different recombinases would not recombine with one another. It should be noted we did observe some unexpected cross-reactivity. In a sense, this is a positive, as it verifies the validity of our measurement construct for testing if two recombinase sites are orthogonal. Indeed, it appears our measurement constructs are more sensitive than those reported in the literature, as cross reactivity for SCre and VCre had not previously been observed [3].

Fig. 1: Basic schematic demonstrating principle of our recombinase measurement constructs. Parallel lines (or capacitor for engineers) represents transcription terminator downstream of a promoter. Triangles of identical colours represent target sites that can recombine with one another. Different coloured triangles are incompatible target sites. Note that RFP reporter gives off quantitative output, so recombination efficiency between two sites can also be quantified.