Difference between revisions of "Part:BBa K2599016"
 
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<partinfo>BBa_K2599016 short</partinfo>
 
<partinfo>BBa_K2599016 short</partinfo>
  
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NCTU_Formosa 2018 designed this sequence to improve the part from NCTU_Formosa 2016.
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<p style="padding-top:20px;font-size:25px"><b>Introduction</b></p>
  
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We changed the general linker to a GS linker to optimize the function of Sf1a.
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A fusion protein is composed by joining of at least two domains that are encoded by separate genes and finally translated as a single polypeptide. And the linker that connects protein domains often plays an important role.
  
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To enhance the function of fusion proteins and provide a proper folding of proteins,
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NCTU_Formosa 2018 modified the linker between Sf1a (spider toxin) and lectin (orally active protein) by elongating short linker AAA (3 a.a.) to GS linker (18 a.a.).
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<p style="padding-top:20px;font-size:25px"><b>Modifying and Improving the Existed Biobrick</b></p>
  
 
===Previous Part:[https://parts.igem.org/Part:BBa_K1974022 (BBa_K1974022)]===
 
===Previous Part:[https://parts.igem.org/Part:BBa_K1974022 (BBa_K1974022)]===
  
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The existing part from NCTU_Formosa 2016 contains the IPTG induced PT7 [https://parts.igem.org/Part:BBa_I712074 (BBa_I712074)], strong ribosome binding site [https://parts.igem.org/Part:BBa_B0034 (BBa_B0034)], Sf1a, AAA linker, snowdrop lectin [https://parts.igem.org/Part:BBa_K1974020 (BBa_K1974020)] and the 6X His-Tag [https://parts.igem.org/Part:BBa_K1223006 (BBa_K1223006)].
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The previous part from NCTU_Formosa 2016 contains the IPTG induced <sub>p</sub>T7 [https://parts.igem.org/Part:BBa_I712074 (BBa_I712074)], strong ribosome binding site [https://parts.igem.org/Part:BBa_B0034 (BBa_B0034)], Sf1a, AAA linker, snowdrop lectin [https://parts.igem.org/Part:BBa_K1974020 (BBa_K1974020)] and the 6X His-Tag [https://parts.igem.org/Part:BBa_K1223006 (BBa_K1223006)].
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{{#tag:html|<img style="width: 60%; padding-left: 18%;" src="https://static.igem.org/mediawiki/2018/b/b4/T--NCTU_Formosa--improve_aaa.png" alt="" />}}
 
{{#tag:html|<img style="width: 60%; padding-left: 18%;" src="https://static.igem.org/mediawiki/2018/b/b4/T--NCTU_Formosa--improve_aaa.png" alt="" />}}
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<div style="width:40%; padding-left: 28%;"><p style="padding-top: 12px; font-size: 10px; text-align: center;"><b>Figure 1.</b> Previous part</p></div>
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<div style="width:40%; padding-left: 28%;"><p style="padding-top: 12px; font-size: 10px; text-align: center;"><b>Figure 1.</b> Previous part Biobrick design</p></div>
  
  
 
===Improvement part===
 
===Improvement part===
  
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In many application, the fusion proteins consisting of multiple protein domains is a popular and highly successful approach to engineering new protein functions. The linker that connects the fusion protein often plays an important role in fusion proteins. Therefore, we decided to change the linker between Sf1a and lectin to try whether the function of this protein become stronger. The GS linker we used in this improvement part was also provided by NCTU_Formosa 2016. It contained 18 amino acid sequence, which is a Gly-Gly-Ser repeated linker.
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The improvement part that NCTU_Formosa 2018 modified contains the IPTG induced <sub>p</sub>T7 [https://parts.igem.org/Part:BBa_I712074 (BBa_I712074)], strong ribosome binding site [https://parts.igem.org/Part:BBa_B0034 (BBa_B0034)], Sf1a, GS linker [https://parts.igem.org/Part:BBa_K1974030 (BBa_K1974030)], snowdrop lectin [https://parts.igem.org/Part:BBa_K1974020 (BBa_K1974020)] and the 6X His-Tag [https://parts.igem.org/Part:BBa_K1223006 (BBa_K1223006)].
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{{#tag:html|<img style="width: 60%; padding-left: 18%;" src="https://static.igem.org/mediawiki/2018/b/bc/T--NCTU_Formosa--mprove_biobrick.png" alt="" />}}
 
{{#tag:html|<img style="width: 60%; padding-left: 18%;" src="https://static.igem.org/mediawiki/2018/b/bc/T--NCTU_Formosa--mprove_biobrick.png" alt="" />}}
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<div style="width:40%; padding-left: 28%;"><p style="padding-top: 12px; font-size: 10px; text-align: center;"><b>Figure 1.</b> Improvement part</p></div>
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<div style="width:40%; padding-left: 28%;"><p style="padding-top: 12px; font-size: 10px; text-align: center;"><b>Figure 2.</b> Improvement part Biobrick design</p></div>
  
  
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<p style="padding-top:20px;font-size:20px"><b>Introduction</b></p>
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<p style="padding-top:20px;font-size:25px"><b>Introduction of μ-segestritoxin-Sf1a and Lectin</b></p>
  
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μ-segestritoxin-Sf1a is kind of insecticidal toxin, contains three disulfide bonds. It will inhibits insect voltage-gated sodium channels by blocking the channel pore.
  
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Lectin is carbohydrate-binding proteins, and is able to bind soluble ectracellular and intercellular glycoproteins.
  
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<p style="padding-top:20px;font-size:25px"><b>Target Insect</b></p>
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{{#tag:html|<img style="width: 40%; padding-left: 27%;" src="https://static.igem.org/mediawiki/2018/8/83/T--NCTU_Formosa--target_insect.png" alt="" />}}
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<div style="width:40%; padding-left: 27%;"><p style="padding-top: 12px; font-size: 10px; text-align: center;"><b>Figure 3.</b>Target Insects</p></div>
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<p style="padding-top:20px;font-size:25px"><b>Experiment</b></p>
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<p style="padding-top:20px;font-size:20px"><b>Preparation of Bio-insecticidal Proteins</b></p>
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We utilized Rosetta-gami DE3 strain to express both the previous part and improvement part. The proteins that produced was then coated on leaves respectively and each leaf was placed inside containers with same number of larvae. The leaf remaining area was observed as shown as below.
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<p style="padding-top:20px;font-size:20px"><b>Result</b></p>
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===Expression===
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===1. Comparison of Plant Protecting Effect of Different Design of Linker between Protein Sf1a and Lectin===
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<br>
  
 
{{#tag:html|<img style="width: 60%; padding-left: 18%;" src="https://static.igem.org/mediawiki/2018/4/4c/T--NCTU_Formosa--sf1a_fig1.png" alt="" />}}
 
{{#tag:html|<img style="width: 60%; padding-left: 18%;" src="https://static.igem.org/mediawiki/2018/4/4c/T--NCTU_Formosa--sf1a_fig1.png" alt="" />}}
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<div style="width:40%; padding-left: 28%;"><p style="padding-top: 12px; font-size: 10px; text-align: center;"><b>Figure 1.</b> Comparison of plant protecting effects</p></div>
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<div style="width:60%; padding-left: 18%;"><p style="padding-top: 12px; font-size: 10px; text-align: center;"><b>Figure 4.</b> Comparison of plant protecting effects by changing the linker between protein Sf1a and lectin. (I): link proteins with GS linker (improvement part BBa_K2599016 ); (II): link proteins with AAA linker (previous part BBa_K1974022); (III): negative control group of Rosetta gami DE3 solution. After feeding for 7 hours, percentage of remained leaf area :  improvement part > previous part > negative control.</p></div>
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<br>
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{{#tag:html|<img style="width: 50%; padding-left: 25%;" src="https://static.igem.org/mediawiki/2018/1/12/T--NCTU_Formosa--improve.png" alt="" />}}
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<div style="width:60%; padding-left: 18%;"><p style="padding-top: 12px; font-size: 10px; text-align: center;"><b>Figure 5.</b> Percentage of remained leaf area of improvement part, previous part and negative control. </p></div>
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<br>
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In conclusion, through comparison, improvement group (I) is more effective in protecting leaf from larvae consuming than previous part (II) and inferred that elongation of linker can enhance the function of fusion proteins and provide a proper folding of proteins.
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From the experiment, we proved the protecting effect of improvement part. Moreover, we observed the abnormal movement of larva in this experiment showing the effect of Sf1a after larva consuming the leaf [https://static.igem.org/mediawiki/2018/5/59/T--NCTU_Formosa--video.mp4 (video of abnormal larva movement)].
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{{#tag:html|<img style="width: 40%; padding-left: 30%;" src="https://static.igem.org/mediawiki/2018/4/41/T--NCTU_Formosa--screenshot.png" alt="" />}}
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<div style="width:28%; padding-left: 35%;"><p style="padding-top: 12px; font-size: 10px; text-align: center;"><b>Figure 6.</b> Screenshot of video</p></div>
  
  

Latest revision as of 09:02, 17 October 2018


T7 Promoter+RBS+Sf1a+GS linker+snowdrop-lectin+linker+6X His-Tag

Introduction

A fusion protein is composed by joining of at least two domains that are encoded by separate genes and finally translated as a single polypeptide. And the linker that connects protein domains often plays an important role.

To enhance the function of fusion proteins and provide a proper folding of proteins, NCTU_Formosa 2018 modified the linker between Sf1a (spider toxin) and lectin (orally active protein) by elongating short linker AAA (3 a.a.) to GS linker (18 a.a.).


Modifying and Improving the Existed Biobrick

Previous Part:(BBa_K1974022)

The previous part from NCTU_Formosa 2016 contains the IPTG induced pT7 (BBa_I712074), strong ribosome binding site (BBa_B0034), Sf1a, AAA linker, snowdrop lectin (BBa_K1974020) and the 6X His-Tag (BBa_K1223006).


Figure 1. Previous part Biobrick design


Improvement part

The improvement part that NCTU_Formosa 2018 modified contains the IPTG induced pT7 (BBa_I712074), strong ribosome binding site (BBa_B0034), Sf1a, GS linker (BBa_K1974030), snowdrop lectin (BBa_K1974020) and the 6X His-Tag (BBa_K1223006).


Figure 2. Improvement part Biobrick design


Introduction of μ-segestritoxin-Sf1a and Lectin

μ-segestritoxin-Sf1a is kind of insecticidal toxin, contains three disulfide bonds. It will inhibits insect voltage-gated sodium channels by blocking the channel pore.

Lectin is carbohydrate-binding proteins, and is able to bind soluble ectracellular and intercellular glycoproteins.


Target Insect

Figure 3.Target Insects

Experiment

Preparation of Bio-insecticidal Proteins

We utilized Rosetta-gami DE3 strain to express both the previous part and improvement part. The proteins that produced was then coated on leaves respectively and each leaf was placed inside containers with same number of larvae. The leaf remaining area was observed as shown as below.


Result

Expression

1. Comparison of Plant Protecting Effect of Different Design of Linker between Protein Sf1a and Lectin


Figure 4. Comparison of plant protecting effects by changing the linker between protein Sf1a and lectin. (I): link proteins with GS linker (improvement part BBa_K2599016 ); (II): link proteins with AAA linker (previous part BBa_K1974022); (III): negative control group of Rosetta gami DE3 solution. After feeding for 7 hours, percentage of remained leaf area : improvement part > previous part > negative control.



Figure 5. Percentage of remained leaf area of improvement part, previous part and negative control.




In conclusion, through comparison, improvement group (I) is more effective in protecting leaf from larvae consuming than previous part (II) and inferred that elongation of linker can enhance the function of fusion proteins and provide a proper folding of proteins.


From the experiment, we proved the protecting effect of improvement part. Moreover, we observed the abnormal movement of larva in this experiment showing the effect of Sf1a after larva consuming the leaf (video of abnormal larva movement).


Figure 6. Screenshot of video


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Reference

1. Elaine Fitches, Martin G. Edwards, Christopher Mee, Eugene Grishin, Angharad M. R. Gatehouse, John P. Edwards, John A. Gatehouse “Fusion proteins containing insect-specific toxins as pest control agents: snowdrop lectin delivers fused insecticidal spider venom toxin to insect haemolymph following oral ingestion,” Journal of Insect Physiology, 2004, 50, pp.61-71
2. Elaine C. Fitches, Prashant Pyati, Glenn F. King, John A. Gatehouse, “ Fusion to Snowdrop Lectin Magnifies the Oral Activity of Insecticidal Omega-Hexatoxin-Hv1a Peptide by Enabling Its Delivery to the Central Nervous System,”
3. Monique J. Windley, Volker Herzig, Slawomir A. Dziemborowicz, Margaret C. Hardy, Glenn F. King and Graham M. Nicholson, “Spider-Venom Peptide as Bioinsecticide,” Toxins Review, 2012, 4, pp. 191-227.
4. A. Lipkin, S. Kozlov, E. Nosyreva, A. Blake, J.D. Windass, E. Grishin (2001, April 9). Novel insecticidal toxins from the venom of the spider Segestria florentina. Toxicon, 40, 125-130.