Difference between revisions of "Part:BBa K1976001"
 
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<a style="color: black" name="&lambda;-Integrase"><h1>&lambda;-Integrase</h1></a>  
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        <a style="color: black" name="&lambda;-Integrase"><h1>&lambda;-Integrase</h1></a>  
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                                <p>The <i>λ</i>‑integrase, originally derived from the <i>λ</i>‑phage, catalyzes the recombination of the phage genome with the chromosomal genome of its host in combination with several assisting proteins. Therefore, two attachment sites are necessary: one located on the bacterial genome (<i>attB</i>) and the other located on the <i>λ</i>‑genome (<i>attP</i>), which also contains several binding sites for regulatory proteins. Having a Tyrosine in its active-site, the &lambda;-integrase belongs to the tyrosine recombinase family.[1]  <br>
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                                    <p>The <i>λ</i>‑integrase, originally derived from the <i>λ</i>‑phage, catalyzes the recombination of the phage genome with the chromosomal genome of its host in combination with several assisting proteins. Therefore, two attachment sites are necessary: one located on the bacterial genome (<i>attB</i>) and the other located on the <i>λ</i>‑genome (<i>attP</i>), which also contains several binding sites for regulatory proteins. Having a Tyrosine in its active-site, the &lambda;-integrase belongs to the tyrosine recombinase family.<sup>1</sup> <br>
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                                </p>
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                            </td>   
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                                <img src="https://static.igem.org/mediawiki/parts/9/95/T--TU_Darmstadt--Integrase_bound_COC%27.png" style="width: 100%" alt="&lambda;-Integrase interacting with DNA">
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                                <div align="center">
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                                    <p >
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                                    <b>Figure 1:</b> Crystal structure of the &lambda;-Integrase in interaction with DNA. Created with VMD (Visual Molecular Dynamics).<a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=1z19"> PDB entry, Biswas, T. <i>et al. 2005</i></a>  
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                                     </p>
 
                                     </p>
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                                 </td>   
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                                    <img src="https://static.igem.org/mediawiki/parts/9/95/T--TU_Darmstadt--Integrase_bound_COC%27.png" style="width: 100%" alt="&lambda;-Integrase interacting with DNA">
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                    </table>
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                                    <div align="center">
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<div id="toc" class="toc"><div id="toctitle"><h2>Contents</h2></div>
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                                        <p >
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<ul>
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                                        <b>Figure 1:</b> Crystal structure of the &lambda;-Integrase in interaction with DNA. Created with VMD (Visual Molecular Dynamics).<a href="http://www.rcsb.org/pdb/explore/explore.do?structureId=1z19"> PDB entry, Biswas, T. <i>et al. 2005</i></a>
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<li class="toclevel-1 tocsection-1"><a href="#&lambda;-Integrase"><span class="tocnumber">1</span> <span class="toctext">&lambda;-Integrase</span></a></li>
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                                        </p>
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<li class="toclevel-1 tocsection-2"><a href="#Recombination Process"><span class="tocnumber">2</span> <span class="toctext">Recombination Process</span></a></li>
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                                    </div>
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<li class="toclevel-1 tocsection-3"><a href="#Usage and Biology"><span class="tocnumber">3</span> <span class="toctext">Usage and Biology</span></a></li>
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                                </td>
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<li class="toclevel-1 tocsection-4"><a href="#Characterisation"><span class="tocnumber">4</span> <span class="toctext">Characterisation</span></a></li>
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                            </tr>     
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<li class="toclevel-1"><a href="#Sequence and Features"><span class="tocnumber">5</span> <span class="toctext">Sequence and Features</span></a></li>
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                        </table>
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</ul>
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        <div id="toc" class="toc"><div id="toctitle"><h2>Contents</h2></div>
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</div>
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            <ul>
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                <li class="toclevel-1 tocsection-1"><a href="#&lambda;-Integrase"><span class="tocnumber">1</span> <span class="toctext">&lambda;-Integrase</span></a></li>
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                <li class="toclevel-1 tocsection-2"><a href="#Recombination Process"><span class="tocnumber">2</span> <span class="toctext">Recombination Process</span></a></li>
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<a name="Recombination Process"><h2>Recombination Process</h2></a>
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                <li class="toclevel-1 tocsection-3"><a href="#Usage and Biology"><span class="tocnumber">3</span> <span class="toctext">Usage and Biology</span></a></li>
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                <li class="toclevel-1"><a href="#Sequence and Features"><span class="tocnumber">5</span> <span class="toctext">Sequence and Features</span></a></li>
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            </ul>
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        </div>
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        <a name="Recombination Process"><h2>Recombination Process</h2></a>
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             <p>The attachment sites contain homologous recognition sequences, called BOB'&nbsp;region (<i>attB</i>) and COC'&nbsp;region (<i>attP</i>). These regions can be connected by the <i>λ</i>‑integrase and the bacterial <i>integration host factor</i> (IHF) via <i>Holliday junction</i>, forming an intasome, a DNA‑protein‑complex, producing hybrid attachment sites <i>attL</i> and <i>attR</i>.</p> <br>
 
             <p>The attachment sites contain homologous recognition sequences, called BOB'&nbsp;region (<i>attB</i>) and COC'&nbsp;region (<i>attP</i>). These regions can be connected by the <i>λ</i>‑integrase and the bacterial <i>integration host factor</i> (IHF) via <i>Holliday junction</i>, forming an intasome, a DNA‑protein‑complex, producing hybrid attachment sites <i>attL</i> and <i>attR</i>.</p> <br>
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                <img src="https://static.igem.org/mediawiki/parts/0/0a/T--TU_Darmstadt--attPrecombination.png" style="width:50%">
 
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                <p align="center"><b>Figure 2:</b>&lambda;-integrase mediated <i>attP/attB</i>-recombination via <i>Holliday junctions</i></p> <br>
 
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            <p>The <i>attP-</i> and <i>attB</i>-strands are nicked by &lambda;-integrases. The active-site amino-acid of the &lambda;-integrase is its <i>Tyrosine 342</i>. The DNA-strands are cleaved via formation of a high energy 3′-phospho-tyrosine intermediate. After cleavage of the two top-strands a four-way <i>Holliday-junction</i> is formed (<b>Figure 2</b>).Here, two free &lambda;-integrase dimers cut the remaining bottom-strands, also via formation of a 3′-phospho-tyrosine intermediate. After the cleavage of the bottom-strands and a rearrangement of the basepairs, the nicked DNA is religated. The whole <i>attp</i>-plasmid is now integrated into the chromosome of the bacterial host. However sequence of the recombination sites <i>attP</i> and <i>attB</i> are not maintained. The integrated DNA is flanked by the two recombined integration-sites <i>attL</i> and <i>attR</i>. The &lambda;-integrase is used by the &lambda;-phage for integration of the phage's genome in context of the lysogenic cycle. (<b>Figure 3</b>)  </p> <br>
 
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                <p align="center"><b>Figure 3:</b> The &lambda;-phage integrates its whole genome via <i>attP/attB</i>-recombination</p>
 
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<a name="Usage and Biology"><h2>Usage and Biology</h2></a>
 
  
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            <p>The &lambda;-Integrase can be used for specific genomic integration of a variable gene of interest (GOI) via <i>attP/attB recombination</i>. For Integration of a certain GOI it has to be cloned into a vector carrying the &lambda;-<i>attP</i>-site. The cloned plasmid together with the presented Part must be transformed into a &lambda;-negative <i>E. Coli</i>-Strain with <i>attB</i>-site.
 
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            The presented &lambda;-Integrase is specific for the &lambda;-attP-site used in <a href="https://parts.igem.org/Part:BBa_K1976000">BBa_K1976000.</a>
 
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<a name="Characterisation"><h2>Characterisation</h2></a>
 
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            <p>
 
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            </p>
 
 
             <center>
 
             <center>
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            <img src="https://static.igem.org/mediawiki/2016/4/47/T--TU_Darmstadt--integrase_nachweis_page_complete.jpg" style="width:20%">
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                <img src="https://static.igem.org/mediawiki/parts/0/0e/DNARecombinationattIntIHF.png" style="width:30%">
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            <p><b>Figure 4:</b> SDS-Page after expression of &lambda;-integrase gene. The gel band of &lambda;-integrase is highlighted by the red circle.</p>
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                <p align="center"><b>Figure 2:</b> &lambda;-integrase mediated <i>attP/attB</i>-recombination.<sup>2</sup> </p> <br>
 
             </center>
 
             </center>
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                <p>The <i>attP-</i> and <i>attB</i>-strands are nicked by &lambda;-integrases. The active-site amino-acid of the &lambda;-integrase is its <i>Tyrosine 342</i>. The DNA-strands are cleaved via formation of a high energy 3′-phospho-tyrosine intermediate. After cleavage of the two top-strands a four-way <i>Holliday-junction</i> is formed.<sup>3</sup> Here, two free &lambda;-integrase dimers cut the remaining bottom-strands, also via formation of a 3′-phospho-tyrosine intermediate. After the cleavage of the bottom-strands and a rearrangement of the basepairs, the nicked DNA is religated. The whole <i>attp</i>-plasmid is now integrated into the chromosome of the bacterial host. However sequence of the recombination sites <i>attP</i> and <i>attB</i> are not maintained. The integrated DNA is flanked by the two recombined integration-sites <i>attL</i> and <i>attR</i>. The &lambda;-integrase is used by the &lambda;-phage for integration of the phage's genome in context of the lysogenic cycle.  </p> <br>
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        <a name="Usage and Biology"><h2>Usage and Biology</h2></a>
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<a name="Sequence and Features"><h2>Sequence and Features</h2></a>
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    <h3>References</h3>
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                <p>The &lambda;-Integrase can be used for specific genomic integration of a variable gene of interest (GOI) via <i>attP/attB recombination</i>. For Integration of a certain GOI it has to be cloned into a vector carrying the &lambda;-<i>attP</i>-site. The cloned plasmid together with the presented Part must be transformed into a &lambda;-negative <i>E. Coli</i>-Strain with <i>attB</i>-site.
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                The presented &lambda;-Integrase is specific for the &lambda;-attP-site used in <a href="https://parts.igem.org/Part:BBa_K1976000">BBa_K1976000.</a>
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                </p>
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        <a name="Sequence and Features"><h2>Sequence and Features</h2></a>
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<partinfo>BBa_K1976001 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K1976001 SequenceAndFeatures</partinfo>
  
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        <h3>References</h3>
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            <li>Tong, W., Warren, D., Seah, N. E., Laxmikanthan, G., Van Duyne, G. D., & Landy, A. (2014). Mapping the λ Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination. Proceedings of the National Academy of Sciences of the United States of America, 111(34), 12366–71. http://doi.org/10.1073/pnas.1413007111</li>
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            <li>https://parts.igem.org/Part:BBa_K1976001</li>
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            <li>Landy, A. (2015). The λ Integrase Site-specific Recombination Pathway, 1–27.</li>
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        </ol>
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<h3>Functional Parameters</h3>
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===Functional Parameters===
 
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<partinfo>BBa_K1976001 parameters</partinfo>
 
<partinfo>BBa_K1976001 parameters</partinfo>

Latest revision as of 17:36, 27 October 2016

λ-Integrase

The λ‑integrase, originally derived from the λ‑phage, catalyzes the recombination of the phage genome with the chromosomal genome of its host in combination with several assisting proteins. Therefore, two attachment sites are necessary: one located on the bacterial genome (attB) and the other located on the λ‑genome (attP), which also contains several binding sites for regulatory proteins. Having a Tyrosine in its active-site, the λ-integrase belongs to the tyrosine recombinase family.1

λ-Integrase interacting with DNA

Figure 1: Crystal structure of the λ-Integrase in interaction with DNA. Created with VMD (Visual Molecular Dynamics). PDB entry, Biswas, T. et al. 2005

Recombination Process

The attachment sites contain homologous recognition sequences, called BOB' region (attB) and COC' region (attP). These regions can be connected by the λ‑integrase and the bacterial integration host factor (IHF) via Holliday junction, forming an intasome, a DNA‑protein‑complex, producing hybrid attachment sites attL and attR.


Figure 2: λ-integrase mediated attP/attB-recombination.2


The attP- and attB-strands are nicked by λ-integrases. The active-site amino-acid of the λ-integrase is its Tyrosine 342. The DNA-strands are cleaved via formation of a high energy 3′-phospho-tyrosine intermediate. After cleavage of the two top-strands a four-way Holliday-junction is formed.3 Here, two free λ-integrase dimers cut the remaining bottom-strands, also via formation of a 3′-phospho-tyrosine intermediate. After the cleavage of the bottom-strands and a rearrangement of the basepairs, the nicked DNA is religated. The whole attp-plasmid is now integrated into the chromosome of the bacterial host. However sequence of the recombination sites attP and attB are not maintained. The integrated DNA is flanked by the two recombined integration-sites attL and attR. The λ-integrase is used by the λ-phage for integration of the phage's genome in context of the lysogenic cycle.


Usage and Biology

The λ-Integrase can be used for specific genomic integration of a variable gene of interest (GOI) via attP/attB recombination. For Integration of a certain GOI it has to be cloned into a vector carrying the λ-attP-site. The cloned plasmid together with the presented Part must be transformed into a λ-negative E. Coli-Strain with attB-site. The presented λ-Integrase is specific for the λ-attP-site used in BBa_K1976000.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]

References

  1. Tong, W., Warren, D., Seah, N. E., Laxmikanthan, G., Van Duyne, G. D., & Landy, A. (2014). Mapping the λ Integrase bridges in the nucleoprotein Holliday junction intermediates of viral integrative and excisive recombination. Proceedings of the National Academy of Sciences of the United States of America, 111(34), 12366–71. http://doi.org/10.1073/pnas.1413007111
  2. https://parts.igem.org/Part:BBa_K1976001
  3. Landy, A. (2015). The λ Integrase Site-specific Recombination Pathway, 1–27.

Functional Parameters

uniprotP03700