Difference between revisions of "Part:BBa K2415000"
 
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<partinfo>BBa_K2415000 short</partinfo>
 
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Mtx 1 was found in Bacillus sphaericus(Bs), an aerobic, Gram-positive, spore-forming bacterium, and it is pathogenic to mosquitoe larvae. Mosquitocidal strains of B. sphaericus can be divided into two groups based on their toxicity to mosquitoe larvae .
 
  
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Three MTX proteins, MTX1, MTX2 and MTX3, were firstly isolated from the low toxicity strains but have also been found in some high-toxicity strains. They are produced in the vegetative phase of growth and are completely degraded in sporulation. MTX has a high toxic level against Culex quinquefasciatus (LC50 ~15 ng ml-1) and Aedes aegypti (LC50 ~50 ng ml-1) larvae.
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Cry4Ba is an 130kDa insecticidal crystal proteins(ICP) that can be isolated from Bacillus thuringiensis subsp. israelensis (Bti). It is specifically toxic against Aedes and Anopheles mosquitoes larvae which are the vectors of Dengue virus, yellow fever virus,  and malaria parasite. The Cry inclusions are solubilized in the midgut lumen of susceptible insects larvae at alkaline pH for both dipterans and lepidopterans, and proteolytically activated by gut proteases to yield the active toxins of ~65-kDa. Subsequently, the activated toxins bind specifically to a variety of receptors such as GPI (glycosylphosphatidyl inositol)-anchored aminopeptidase-N and GPI-anchored alkaline phosphatase that are found on the brush-border membrane (BBM) of midgut epithelial cells. This toxin-receptor interaction can assist toxin penetration into the cell membrane, leading to the formation of ion-permeable pores and eventually causeing osmotic lysis of target cells.
  
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The mosquitocidal toxin (MTX) from Bacillus sphaericus(Bs) is a member of ADP ribosyltransferases family . Mtx1 is a ~100 kDa soluble protein, consisting of two parts including the N-terminal 27 kDa domain containing ADP-ribosylase activity and the C-terminal 70 kDa domain which is very similar to the carbohydrate-binding protein. The mature MTX (without the putative signal sequence) is processed into a 27 kDa N-terminal fragment and a 70 kDa C-terminal fragment by trypsin. The N-terminal ADP ribosyl moiety connects to protein amino acid residues in the intestinal cells of mosquito larvae, and thus the Mtx1 disable the proteins and kill the cell.
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Sporulating cells of Bacillus thuringiensis (Bti) can produce insecticidal crystal proteins, or ICPs. The ICPs are classified by amino acid sequence homology into two main families, the Cry and the Cyt toxins. Cry insecticidal proteins, which are known as δ-endotoxins, are produced by the Gram-positive soil bacterium Bacillus thuringiensis (Bti) produced in the form of crystalline inclusions during sporulation. These crystal toxins are cytolytic pore-forming toxins that are lethal to various insect larvae.
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The structure of Cry toxin shows a common topology composed of three domains. Domain I is an a-helix bundle, which is believed to be responsible for pore formation on the basis of its structural characteristics. Domain II is a b-prism of three antiparallel sheets with loops ending at its apex, which is thought to be involved in receptor binding. Domain III is a lectin-like b-sandwich, which is important to the stability of the toxin, and has been found to participate in determining toxin specificity.  
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The following picture shows the amplification of the gene cry4Ba by PCR. (Left lane,about 3.5kb)
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                                        https://static.igem.org/mediawiki/2017/a/a7/T--GZHS-United--result2.png
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The following picture shows expression and purification of Cry4Ba. Lane 1 is our negative control containing bacterial protein of E. coli BL21, lane 2 is our target protein before purification containing bacterial protein of Cry4Ba E. coli BL21, lane 3 is the protein Cry4Ba after purification. It can be seen from the graph that we have successfully obtained the target protein with high purity.
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                                        https://static.igem.org/mediawiki/2017/7/72/T--GZHS-United--result4.png
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We used Cry4Ba E.coli BL21 to do a toxicity test to find out the suitable concentration of killing Aedes larvae. According to the figure below, there is an obvious trend that experimental groups with higher concentrations of Cry4Ba E.coli have higher lethality rates.
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        https://static.igem.org/mediawiki/2017/7/76/T--GZHS-United--demonstrate2.png
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 09:49, 27 October 2017


Cry4Ba

Cry4Ba is an 130kDa insecticidal crystal proteins(ICP) that can be isolated from Bacillus thuringiensis subsp. israelensis (Bti). It is specifically toxic against Aedes and Anopheles mosquitoes larvae which are the vectors of Dengue virus, yellow fever virus, and malaria parasite. The Cry inclusions are solubilized in the midgut lumen of susceptible insects larvae at alkaline pH for both dipterans and lepidopterans, and proteolytically activated by gut proteases to yield the active toxins of ~65-kDa. Subsequently, the activated toxins bind specifically to a variety of receptors such as GPI (glycosylphosphatidyl inositol)-anchored aminopeptidase-N and GPI-anchored alkaline phosphatase that are found on the brush-border membrane (BBM) of midgut epithelial cells. This toxin-receptor interaction can assist toxin penetration into the cell membrane, leading to the formation of ion-permeable pores and eventually causeing osmotic lysis of target cells.

Sporulating cells of Bacillus thuringiensis (Bti) can produce insecticidal crystal proteins, or ICPs. The ICPs are classified by amino acid sequence homology into two main families, the Cry and the Cyt toxins. Cry insecticidal proteins, which are known as δ-endotoxins, are produced by the Gram-positive soil bacterium Bacillus thuringiensis (Bti) produced in the form of crystalline inclusions during sporulation. These crystal toxins are cytolytic pore-forming toxins that are lethal to various insect larvae.

The structure of Cry toxin shows a common topology composed of three domains. Domain I is an a-helix bundle, which is believed to be responsible for pore formation on the basis of its structural characteristics. Domain II is a b-prism of three antiparallel sheets with loops ending at its apex, which is thought to be involved in receptor binding. Domain III is a lectin-like b-sandwich, which is important to the stability of the toxin, and has been found to participate in determining toxin specificity.

The following picture shows the amplification of the gene cry4Ba by PCR. (Left lane,about 3.5kb) 

                                       T--GZHS-United--result2.png

The following picture shows expression and purification of Cry4Ba. Lane 1 is our negative control containing bacterial protein of E. coli BL21, lane 2 is our target protein before purification containing bacterial protein of Cry4Ba E. coli BL21, lane 3 is the protein Cry4Ba after purification. It can be seen from the graph that we have successfully obtained the target protein with high purity. 

                                       T--GZHS-United--result4.png

We used Cry4Ba E.coli BL21 to do a toxicity test to find out the suitable concentration of killing Aedes larvae. According to the figure below, there is an obvious trend that experimental groups with higher concentrations of Cry4Ba E.coli have higher lethality rates. 

        T--GZHS-United--demonstrate2.png

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 69
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 2493
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 475