Difference between revisions of "Part:BBa K2599016"

Revision as of 07:21, 16 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

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

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

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.(I): Fusion proteins with GS linker (Improvement part BBa_K2599016 ); (II): Fusion proteins with AAA linker (Previous part BBa_K1974022); (III): Negative control group of Rosetta gami DE3 solution. After feeding for 7 hours, leaf area of (I) remained unchanged compared with leaf remaining area after 3 hours; While leaf of (II) and (III) was continued consumed by larva. Through comparison, improvement group (I) is more effective in protecting leaf from larvae consuming than previous part (II).

2. Plant Protecting Effect of sequence Sf1a-GS Linker-Lectin (Improvement Part) in pET-32a

After preliminary testing the plant protecting effect of improvement part, we changed the vector of insert gene from pSB1A3 into pET-32a for higher expression of peptide sequences.

Figure 4. Plant protecting effects by inserting the improvement part into pET-32a.

From the experiment, we again 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).

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.

@@ Line 16: / Line 16: @@
 ===Previous Part:[https://parts.igem.org/Part:BBa_K1974022 (BBa_K1974022)]===
-The existing 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)].
+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)].
@@ Line 26: / Line 26: @@
 ===Improvement part===
-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. We utilized this linker to optimize the fuction of Sf1a and lectin.
+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)].
@@ Line 34: / Line 34: @@
-<p style="padding-top:20px;font-size:20px"><b>Introduction</b></p>
+<p style="padding-top:20px;font-size:25px"><b>Introduction of μ-segestritoxin-Sf1a and Lectin</b></p>
 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.
@@ Line 41: / Line 41: @@
-===Target Insect===
+<p style="padding-top:20px;font-size:25px"><b>Target Insect</b></p>
-<p style="padding-top:20px;font-size:20px"><b>Result</b></p>
+<p style="padding-top:20px;font-size:25px"><b>Experiment</b></p>
+<p style="padding-top:20px;font-size:18px"><b>Preparation of Bio-insecticidal Proteins</b></p>
+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.
+<p style="padding-top:20px;font-size:18px"><b>Result</b></p>
+===Expression===
 ===1. Comparison of Plant Protecting Effect of Different Design of Linker between Protein Sf1a and Lectin===
@@ Line 53: / Line 63: @@
 {{#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="" />}}
-<div style="width:40%; padding-left: 28%;"><p style="padding-top: 12px; font-size: 10px; text-align: center;"><b>Figure 4.</b> Comparison of plant protecting effects</p></div>
+<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.(I): Fusion proteins with GS linker (Improvement part BBa_K2599016 ); (II): Fusion proteins with AAA linker (Previous part BBa_K1974022); (III): Negative control group of Rosetta gami DE3 solution. After feeding for 7 hours, leaf area of (I) remained unchanged compared with leaf remaining area after 3 hours; While leaf of (II) and (III) was continued consumed by larva. Through comparison, improvement group (I) is more effective in protecting leaf from larvae consuming than previous part (II).</p></div>
 <br>
-(I): Fusion proteins with GS linker (Improvement part BBa_K2599016 ); (II): Fusion proteins with AAA linker (Previous part BBa_K1974022); (III): Negative control group of Rosetta gami DE3 solution. After feeding for 7 hours, leaf area of (I) remained unchanged compared with leaf remaining area after 3 hours; While leaf of (II) and (III) was continued consumed by larva. Through comparison, improvement group (I) is more effective in protecting leaf from larvae consuming than previous part (II).
@@ Line 62: / Line 70: @@
 ===2. Plant Protecting Effect of sequence Sf1a-GS Linker-Lectin (Improvement Part) in pET-32a===
-<br>
+After preliminary testing the plant protecting effect of improvement part, we changed the vector of insert gene from pSB1A3 into pET-32a for higher expression of peptide sequences.
 {{#tag:html|<img style="width: 75%; padding-left: 10%;" src="https://static.igem.org/mediawiki/2018/9/9c/T--NCTU_Formosa--sf1a_fig2-1.png" alt="" />}}
@@ Line 70: / Line 78: @@
 <br>
-Through comparison, improvement part with the highest concentration shown the most effective as it had the largest leaf area remaining.
+From the experiment, we again 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).