Difference between revisions of "Team:NYMU-Taipei/Design"

Line 53: Line 53:
 
<b style="font-size:16px;">What is KillerRed ?</b>
 
<b style="font-size:16px;">What is KillerRed ?</b>
  
−
<p style="font-size:16px;">The phototoxic KillerRed protein is a red fluorescent protein with excitation and emission maxima at 585 and 610 nm respectively. When the protein comes in contact with light of wavelengths of 520-590 nm, it produces reactive oxygen species (ROS) along with intense photobleaching. The phototoxicity induced by KillerRed generated ROS is 1,000times greater than those produced by common fluorescent proteins5. The increase concentrations of ROS in the cytoplasm disrupt normal cellular functions and impede fungal growth and, in the best case scenario, induce programed necrosis6.</p>
+
<p style="font-size:16px;">The KillerRed protein is a red fluorescent protein with excitation and emission maxima at 585 and 610 nm respectively. It is engineered from anm2CP to be phototoxic.<sup>[3]</sup> When the protein comes in contact with light of wavelengths of 520-590 nm, it produces reactive oxygen species (ROS) along with intense photobleaching. The phototoxicity induced by KillerRed generated ROS is 1,000times greater than those produced by common fluorescent proteins<sup>[5]</sup>.  Expression of KillerRed and irradiation with light may act a kill-switch for biosafety applications<sup>[3]</sup>. The increase concentrations of ROS in the cytoplasm disrupt normal cellular functions and impede fungal growth and, in the best case scenario, induce programed necrosis<sup>[6]</sup>.</p>
  
 
<img src="https://static.igem.org/mediawiki/2016/1/1e/T-NYMU-Taipei-photo-14725328_901026143361261_1257045046_o.png" width="100%" />
 
<img src="https://static.igem.org/mediawiki/2016/1/1e/T-NYMU-Taipei-photo-14725328_901026143361261_1257045046_o.png" width="100%" />
Line 79: Line 79:
  
 
[1] Review on safety of the entomopathogenic fungus Metarhizium anisopliae
 
[1] Review on safety of the entomopathogenic fungus Metarhizium anisopliae
 +
[2]Wang, C., & St. Leger, R. J. (2006). A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses. Proceedings of the National Academy of Sciences of the United States of America, 103, 6647-6652.
 +
 +
[3]2013 Carnegie_Mellon ;https://2013.igem.org/Team:Carnegie_Mellon/KillerRed
 +
 
<li style="list-style-type:decimal;"><p style="font-size:16px;">Sudakin D.L. Biopesticides. Toxicol. Rev. 2003;22:83–90. doi: 10.2165/00139709-200322020-00003.</p></li>
 
<li style="list-style-type:decimal;"><p style="font-size:16px;">Sudakin D.L. Biopesticides. Toxicol. Rev. 2003;22:83–90. doi: 10.2165/00139709-200322020-00003.</p></li>
 
<li style="list-style-type:decimal;"><p style="font-size:16px;">St Leger, R., Joshi, L., Bidochka, M. J., & Roberts, D. W. (1996). Construction of an improved mycoinsecticide overexpressing a toxic protease. Proceedings of the National Academy of Sciences of the United States of America, 93(13), 6349–6354.</p></li>
 
<li style="list-style-type:decimal;"><p style="font-size:16px;">St Leger, R., Joshi, L., Bidochka, M. J., & Roberts, D. W. (1996). Construction of an improved mycoinsecticide overexpressing a toxic protease. Proceedings of the National Academy of Sciences of the United States of America, 93(13), 6349–6354.</p></li>

Revision as of 22:16, 19 October 2016

Overview

Through the utilization of different gene expression and location of M. anisopliae cells during different stages of its infection cycle alongside with light-induced phototoxic fluorescent proteins, we aim to design entomopathogenic-fungi-specific killswitch that can greatly mitigate the safety concerns of the genetic modified fungal insecticides in hope that it will increase the feasibility of wide spread use of these enhanced bioinsecticides.

Design


What is the infectioni cycle of M anisopliae ?

Here we show a short overview: The infection process of M. anisopliae is similar to other entomopathogenic fungi, i.e. the infection pathway consists on the following steps: (1) attachment of the spore to the cuticle, (2) germination and formation of appressoria, (3) penetration through the cuticle, (4) overcoming of the host response and immune defence reactions of the host, (5) spreading within the host by formation of hyphal bodies or blastospores, i.e. yeast like cells, and (6) outgrowing the dead host and production of new conidia.[1]

What is PMcl1 ?

Metarhizium-collogen-like promoter, or PMcl1, is a strong insect-hemolymph inducible promoter. This promoter controls the production of Metarhizium-collagen-like-proteins(3) in wildtype Metarhizium anisopliae, the transcripts of the Mcl1 gene could be detected within 20 minutes of the fungus contacting the hemolymph, but could not be detected when the fungus is cultured in any other medium(4). Utilizing the promoter of PMcl1 in genetically engineering Metarhizium allows the expression of target genes to be limited to the hemocoel of the fungus' host insects, ensuring the specificity of gene expression[2].

What is KillerRed ?

The KillerRed protein is a red fluorescent protein with excitation and emission maxima at 585 and 610 nm respectively. It is engineered from anm2CP to be phototoxic.[3] When the protein comes in contact with light of wavelengths of 520-590 nm, it produces reactive oxygen species (ROS) along with intense photobleaching. The phototoxicity induced by KillerRed generated ROS is 1,000times greater than those produced by common fluorescent proteins[5]. Expression of KillerRed and irradiation with light may act a kill-switch for biosafety applications[3]. The increase concentrations of ROS in the cytoplasm disrupt normal cellular functions and impede fungal growth and, in the best case scenario, induce programed necrosis[6].

Circuit


Our Killswitch Circuit:

The killswitch circuit contains the hemolymph-induced promoter, PMcl, followed by the KillerRed gene (BBa_K1184000) and a trpC terminator, TtrpC. This sets the mass production of KillerRed proteins to the hemolymph infection phase of M. anisopliae’s life cycle.



____

Reference

[1] Review on safety of the entomopathogenic fungus Metarhizium anisopliae [2]Wang, C., & St. Leger, R. J. (2006). A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses. Proceedings of the National Academy of Sciences of the United States of America, 103, 6647-6652. [3]2013 Carnegie_Mellon ;https://2013.igem.org/Team:Carnegie_Mellon/KillerRed

  • Sudakin D.L. Biopesticides. Toxicol. Rev. 2003;22:83–90. doi: 10.2165/00139709-200322020-00003.

  • St Leger, R., Joshi, L., Bidochka, M. J., & Roberts, D. W. (1996). Construction of an improved mycoinsecticide overexpressing a toxic protease. Proceedings of the National Academy of Sciences of the United States of America, 93(13), 6349–6354.

  • Wang CS, St Leger RJ (2007) A scorpion neurotoxin increases the potency of a fungal insecticide. Nat Biotechnol 25: 1455–1456.

  • Wang, C., & St. Leger, R. J. (2006). A collagenous protective coat enables Metarhizium anisopliae to evade insect immune responses. Proceedings of the National Academy of Sciences of the United States of America, 103(17), 6647–6652.

  • Pletnev, S., Gurskaya, N. G., Pletneva, N. V., Lukyanov, K. A., Chudakov, D. M., Martynov, V. I., … Pletnev, V. (2009). Structural Basis for Phototoxicity of the Genetically Encoded Photosensitizer KillerRed. The Journal of Biological Chemistry, 284(46), 32028–32039.

  • Breitenbach, M., Weber, M., Rinnerthaler, M., Karl, T., & Breitenbach-Koller, L. (2015). Oxidative Stress in Fungi: Its Function in Signal Transduction, Interaction with Plant Hosts, and Lignocellulose Degradation. Biomolecules,5(2), 318–342.