Difference between revisions of "Part:BBa K2609026"
 
(8 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K2609026 short</partinfo>
 
<partinfo>BBa_K2609026 short</partinfo>
  
−
This part generates the three Lambda ReD recombinases - exo, gam, beta - when expressed in a T7 expression strain like E. coli BL21(DE3).
+
This part generates the three Lambda RED recombinases - exo, gam, beta - when expressed in a T7 expression strain like E. coli BL21(DE3).  
−
 
+
<h4>Sequence and Features</h4>
 +
<partinfo>BBa_K2609026 SequenceAndFeatures</partinfo>
 
===Usage and Biology===
 
===Usage and Biology===
 
<html>
 
<html>
Line 17: Line 18:
 
<h3> IISc-Bangalore 2018</h3>
 
<h3> IISc-Bangalore 2018</h3>
 
<p>
 
<p>
−
     We used the this Lambda RED recombination system with a T4 Endolysin gene (<a href="https://parts.igem.org/Part:BBa_K2609017">BBa_K2609017</a>) to recombine Bacteriophage T4 and create a lysis deficient phage. This lysis deficient phage triggers its host to produce a monocyte chemoattractant <i>mcp-1</i> (<a href="https://parts.igem.org/Part:BBa_K2609000">BBa_K2609000</a>).
+
     For their Phage Assisted Imune Recruitment (<a href="http://2018.igem.org/Team:IISc-Bangalore/PAIR">PAIR</a>) the IISc 2018 iGEM Team used this part to recombine the Bacteriophage T4 genome to produce a lysis deficient phage. This was done using the parts <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609008">BBa_K2609008</a> and <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K2609009">BBa_K2609009</a> which have flanking sequences homologous to the flanking regions of Endolysin gene (e gene) on the T4 genome. A recombinant T4 Endolysin (<a href="https://parts.igem.org/Part:BBa_K2609017">BBa_K2609017</a>) was used in conjuction with this part to screen for recombinants. Following successful recombination, their lysis deficient phage would trigger it's host to secrete mcp-1 (<a href="https://parts.igem.org/Part:BBa_K2609000">BBa_K2609000</a>).
 
</p>
 
</p>
 +
<h2> Characterisation</h2>
 +
<div style="height: auto">
 +
    <h3>IISc-Bangalore 2018</h3>
 +
    <h4>Expression</h4>
 +
    <p>
 +
        The part was transformed into E. coli BL21(DE3) since it contains the T7 polymerase under a lac promoter. It was induced with IPTG in mid log phase and the cell pellet was loaded onto an SDS PAGE. The following are the exact conditions:
 +
    </p>
 +
    <figure style="float:right; width:35%; text-align: left; font-style: italic; font-size: smaller; text-indent: 0; border: thin silver solid; margin: 0.5em; padding: 0.5em;">
 +
        <img src="https://static.igem.org/mediawiki/parts/d/d7/T--IISc-Bangalore--lambdaT7exp.png" style="width: 100%; border: 1px solid black;">
 +
        <figcaption>Following growth and induction, 1mL of the culture was pelleted down and resuspended in 100μL of dH<sub>2</sub>O and mixed with 100μL of SDS sample buffer. 10μL of this was loaded onto a 12% acrylamide SDS PAGE.</figcaption>
 +
    </figure>
 +
    <ul>
 +
        <li>Initial Growth Temperature: 37°C</li>
 +
        <li>Growth Medium: LB</li>
 +
        <li>Chloramphenicol Concentration: 35μg/mL</li>
 +
        <li>Induction OD600: 0.6</li>
 +
        <li>IPTG Concentration: 500μM</li>
 +
        <li>Growth Temperature Following Induction: 37°C</li>
 +
        <li>Growth Time Following Induction: 3hrs</li>
 +
    </ul>
 +
    <p>
 +
        Bands were observed slightly below 32kDa ladder band and at the 25kDa ladder band. These two correspond to the bet protein (29.7 kDa) and the exo protein (25.9 kDa). The other protein - gam (16.3 kDa) - cannot be seen on the gel. This could be because the part has only one RBS sequence for all three proteins. It could be either that the third protein is being produced in very small amounts or not at all.
 +
    </p>
 +
</div>
 
</body>
 
</body>
 
</html>
 
</html>
 
<!-- -->
 
<!-- -->
−
<span class='h3bb'>Sequence and Features</span>
+
 
−
<partinfo>BBa_K2609026 SequenceAndFeatures</partinfo>
+
 
===References===
 
===References===
 
[1] Mosberg, J. A., Lajoie, M. J., & Church, G. M. (2010). Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate. Genetics, 186(3), 791–799. http://doi.org/10.1534/genetics.110.120782
 
[1] Mosberg, J. A., Lajoie, M. J., & Church, G. M. (2010). Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate. Genetics, 186(3), 791–799. http://doi.org/10.1534/genetics.110.120782
Line 30: Line 54:
  
 
[3]Datsenko K A, Wanner B L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proceedings of the National Academy of Sciences, 2000, 97(12): 6640-6645.
 
[3]Datsenko K A, Wanner B L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proceedings of the National Academy of Sciences, 2000, 97(12): 6640-6645.
 +
 +
[4]https://www.uniprot.org/uniprot/P03697
 +
 +
[5]https://www.uniprot.org/uniprot/P03698
 +
 +
[6]https://www.uniprot.org/uniprot/P03702
 +
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 22:56, 17 October 2018


Lambda Red Recombinases under T7 expression system

This part generates the three Lambda RED recombinases - exo, gam, beta - when expressed in a T7 expression strain like E. coli BL21(DE3).

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1943
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1460
  • 1000
    COMPATIBLE WITH RFC[1000]

Usage and Biology

Biology

The genes exo, gam and beta are a part of Bacteriophage Lambda's genome and is used in recomibination of bacteriophage DNA. The gam protein binds to the RecBCD nuclease of the host, thus protecting linear viral DNA from degradation. The protein exo is a 5' to 3' exonuclease which exposes the ends of linear dsDNA. The protein beta promotes single strand annealing and hence promotes homologous recombination. It is also important in rollin circle DNA replication which comes late in the infective cycle of the lambda phage.

Usage

IISc-Bangalore 2018

For their Phage Assisted Imune Recruitment (PAIR) the IISc 2018 iGEM Team used this part to recombine the Bacteriophage T4 genome to produce a lysis deficient phage. This was done using the parts BBa_K2609008 and BBa_K2609009 which have flanking sequences homologous to the flanking regions of Endolysin gene (e gene) on the T4 genome. A recombinant T4 Endolysin (BBa_K2609017) was used in conjuction with this part to screen for recombinants. Following successful recombination, their lysis deficient phage would trigger it's host to secrete mcp-1 (BBa_K2609000).

Characterisation

IISc-Bangalore 2018

Expression

The part was transformed into E. coli BL21(DE3) since it contains the T7 polymerase under a lac promoter. It was induced with IPTG in mid log phase and the cell pellet was loaded onto an SDS PAGE. The following are the exact conditions:

Following growth and induction, 1mL of the culture was pelleted down and resuspended in 100μL of dH2O and mixed with 100μL of SDS sample buffer. 10μL of this was loaded onto a 12% acrylamide SDS PAGE.
  • Initial Growth Temperature: 37°C
  • Growth Medium: LB
  • Chloramphenicol Concentration: 35μg/mL
  • Induction OD600: 0.6
  • IPTG Concentration: 500μM
  • Growth Temperature Following Induction: 37°C
  • Growth Time Following Induction: 3hrs

Bands were observed slightly below 32kDa ladder band and at the 25kDa ladder band. These two correspond to the bet protein (29.7 kDa) and the exo protein (25.9 kDa). The other protein - gam (16.3 kDa) - cannot be seen on the gel. This could be because the part has only one RBS sequence for all three proteins. It could be either that the third protein is being produced in very small amounts or not at all.

References

[1] Mosberg, J. A., Lajoie, M. J., & Church, G. M. (2010). Lambda Red Recombineering in Escherichia coli Occurs Through a Fully Single-Stranded Intermediate. Genetics, 186(3), 791–799. http://doi.org/10.1534/genetics.110.120782

[2]Sharan S K, Thomason L C, Kuznetsov S G, et al. Recombineering: a homologous recombination-based method of genetic engineering[J]. Nature protocols, 2009, 4(2): 206-223.

[3]Datsenko K A, Wanner B L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[J]. Proceedings of the National Academy of Sciences, 2000, 97(12): 6640-6645.

[4]https://www.uniprot.org/uniprot/P03697

[5]https://www.uniprot.org/uniprot/P03698

[6]https://www.uniprot.org/uniprot/P03702