Part:BBa_K2599016
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE 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.
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