Difference between revisions of "Part:BBa K1974012"
 
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<h1>'''Introduction:'''</h1>
 
<h1>'''Introduction:'''</h1>
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[[File:NCTU FORMOSA S1.png|800px|thumb|center|'''Figure 1.''' P<sub>T7</sub>+RBS+Sf1a+linker+His-Tag+terminator ]]
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By ligating the IPTG induced P<sub>T7</sub>(BBa_ I712074), strong ribosome binding site (BBa_B0034), sf1a, linker, and the 6X His-Tag (BBa_ K1223006), we can express Sf1a, the toxin by IPTG induction
 +
.<br>
 +
This year we create a revolutionary system that integrates biological pesticides, an automatic detector, a sprinkler, and IoT. We made a database that contains most of the spider toxins and selected the target toxins by programming. μ2-segestritoxin-Sf1a is coded for the venom of a spider, <i>Segestria florentine</i>. It is under the control of the strong P<sub>T7</sub>. A 6X His-Tag is added for further protein purification.
  
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<p style="padding-top:20px;font-size:20px"><b>Mechanism of Sf1a</b></p>
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μ2-segestritoxin-Sf1a has a structure called ICK(inhibitor cysteine knot). This kind of structure contains four disulfide bonds. With this structure, Sf1a can resist the high temperature, acid-base solution and the digest juice of insect gut. Sf1a can bind on insect voltage-gated sodium channel Site-1, making it paralyze and die eventually.<sup>[1]</sup>
  
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<!--圖*2-->
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<p style="padding-top:20px;font-size:20px"><b>Features of Sf1a</b></p>
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<p style="padding:1px;font-size:16px"><b>1. Non-toxic</b></p>μ2-segestritoxin-Sf1a is non-toxic to mammals. Since the structure of the target ion channel is different, μ2-segestritoxin-Sf1a does not harm mammals. So it is safe to use it as a biological pesticide.<sup>[2]</sup>
 +
<br>
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<p style="padding:1px;font-size:16px"><b>2. Biodegradable</b>T</p>he toxin is a peptide, so it must degrade over time. After degradation, the toxin will become nutrition in the soil.
 +
<br>
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<p style="padding:1px;font-size:16px"><b>3. Species-specific</b></p>According to reference, μ2-segestritoxin-Sf1a has specificity to Lepidopteran (moths) and Dipteran (flies).
 +
<br>
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<p style="padding:1px;font-size:16px"><b>4. Eco-friendly</b></p>Compare with a chemical pesticide, μ2-segestritoxin-Sf1a will not remain in soil and water so that it will not pollute the environment and won’t harm the ecosystem.
  
  
 +
Together, using Sf1a is totally an environmentally friendly way for solving harmful insect problems by using this ion channel inhibitor as a biological pesticide.
  
 +
<p style="padding-top:20px;font-size:20px"><b>Target insect</b></p>
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[[File:NCTU FORMOSA Sf1a-1.png|800px|thumb|center|'''Figure2''' Target insects]]
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<!--target picture-->
  
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<h1>'''Experiment'''</h1>
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<p style="padding:1px;font-size:16px"><b>1. Cloning </b></p><br>After assembling the DNA sequences from the basic parts, we recombined each P<sub>T7</sub>+B0034+Sf1a +linker+6X His-Tag  gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each part. The DNA sequence length of these parts is around 250-300 b.p. In this PCR experiment, the toxin product's size should be near at 550-600 b.p. 
 +
[[File:Sf1a cloning results.jpg|200px|thumb|center|'''Figure 3.''' P<sub>T7</sub> + RBS + Sf1a+linker+6X His-Tag <br>
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The DNA sequence length of P<sub>T7</sub> + RBS + Sf1a+linker+6X His-Tag is around 250-300 b.p. In this PCR experiment, the product’s size should be close to 550-600 b.p.
 +
]]
 +
 +
<p style="padding:1px;font-size:16px"><b>2. Expressing</b></p><br>We chose <i>E.coli</i> Rosetta gami strain, which can form the disulfide bonds in the cytoplasm to express the protein. To verify the <i>E.coli</i> express the P<sub>T7</sub> + RBS + Sf1a+linker+6X His-Tag which contains disulfide bonds, we treated the sample in two different ways. A means adding β-mercaptoethanol and sample buffer. β-mercaptoethanol can break the disulfide bonds of Sf1a and make it a linear form.
 +
The other one adding sample buffer is the native form of P<sub>T7</sub> + RBS + Sf1a+linker+6X His-Tag which maintains its structure. B is adding only sample buffer. The two samples are treated in boiling water for 15 mins.
 +
The SDS-PAGE shows that the native P<sub>T7</sub> + RBS + Sf1a+linker+6X His-Tag is smaller than linear one because the disulfide bonds in P<sub>T7</sub> + RBS + Sf1a+linker+6X His-Tag make the whole structure a globular shape.
 +
[[File:NCTU _S-express.jpg|300px|thumb|center|'''Figure 4.''' P<sub>T7</sub> + RBS + Sf1a+linker+6X His-Tag (control: Without constructed plasmid)
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<br>We can see the band of Sf1a at 5-6 kDa.
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<br>A: add β-mercaptoethanol and sample buffer
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<br>B: add sample bufferF
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]]
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<p style="padding:1px;font-size:16px"><b>3. Purification</b></p><br>We sonicated the bacteria and purified the protein by 6X His-Tag behind the peptide using Nickel resin column. Then we ran the SDS-PAGE to verify the purification and analyze the concentration of Sf1a.
 +
[[File:NCTU _S-purify.jpg|300px|thumb|center|'''Figure 5.''' Protein electrophoresis of Sf1a-6X His-Tag purification<br>
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A is the sonication product.<br>B is the elution product of purification
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]]
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<!--Also, we do the UV test and model the degradation rate.-->
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<p style="padding:1px;font-size:16px"><b>4.Modeling</b></p><br>According to reference, the energy of Ultraviolet will break the disulfide bonds and the toxicity is also decreased. To take the parameter into consideration for our automatic system, we modeled the degradation rate of the protein and modified the program in our device.Therefore PANTIDE was be test under the Ultraviolet and model the degradation rate. the Protein Electrophoresis was showed below.
 +
[[File:Sf1a_degradation_SDS.PNG|400px|thumb|center|'''Figure 6.''' SDS-PAGE gel and the concentrations of UV radiolytic oxidation test to native μ-segestritoxin-Sf1a (Sf1a, 6.2 kDa). The samples are marked on the top of gel.]]
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<!--放濃度對時間作圖--><!---預測降解速率的圖------>
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<p style="padding:1px;font-size:16px"><b>5. Device</b></p><br>We designed a device that contains a detector, a sprinkler, and an integrated hardware with users by APP through IoT talk. We use an infrared detector to detect the number of the pest and predict what time to spray the farmland. Furthermore, other detectors like temperature, humidity, lamination, pressure of carbon dioxide are also installed in our device. At the same time, the APP that displays all the information about the farmland would contact the users and spray biological pesticides automatically. This device can make farmers control the farmland remotely.
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<br>
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<H1>'''Results'''</H1>
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Pantide-expressed E. coli Rosetta gami strain and diluted it with the three concentration.We applied the sample onto the leaf disks and put five cutworms into the separate cabinets for feeding assays. The positive control in the experiment was to apply <i>Bacillus thuringiensis</i>, which is the most widely-used bioinsecticide. We preserved all the result of the remained leaves sealing with the glass paper and calculated the ratio of the remained area on the leaves. The collected data were analyzed by t – test. Here are the feeding assay results.
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[[File:NCTU_DOSE_S_2.png|400px|thumb|center|'''Figure 7.''' Above is leaves remaining area of Negative control ( DDH<sub>2</sub>O), Positive control (<i> Bacillus thuringiensis  </i> bacteria ), Sf1a+linker+6XHis-Tag, Sf1a+linker+snowdrop-lectin+linker+6XHis-Tag]]
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[[File:NCTU_leaves_s_1.png|400px|thumb|center|'''Figure 8.''' Above are leaves with of Negative control ( DDH<sub>2</sub>O), Positive control ( <i>Bacillus thuringiensis </i>bacteria ), Sf1a+linker+6X His-Tag]]
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 +
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<h1>'''Reference'''</h1>
 +
1. 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.
 +
<br>
 +
2. 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
  
 
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Latest revision as of 23:07, 30 October 2016

T7Promoter+RBS+Sf1a+linker+6X His-Tag

Introduction:

Figure 1. PT7+RBS+Sf1a+linker+His-Tag+terminator

By ligating the IPTG induced PT7(BBa_ I712074), strong ribosome binding site (BBa_B0034), sf1a, linker, and the 6X His-Tag (BBa_ K1223006), we can express Sf1a, the toxin by IPTG induction .
This year we create a revolutionary system that integrates biological pesticides, an automatic detector, a sprinkler, and IoT. We made a database that contains most of the spider toxins and selected the target toxins by programming. μ2-segestritoxin-Sf1a is coded for the venom of a spider, Segestria florentine. It is under the control of the strong PT7. A 6X His-Tag is added for further protein purification.

Mechanism of Sf1a

μ2-segestritoxin-Sf1a has a structure called ICK(inhibitor cysteine knot). This kind of structure contains four disulfide bonds. With this structure, Sf1a can resist the high temperature, acid-base solution and the digest juice of insect gut. Sf1a can bind on insect voltage-gated sodium channel Site-1, making it paralyze and die eventually.[1]

Features of Sf1a

1. Non-toxic

μ2-segestritoxin-Sf1a is non-toxic to mammals. Since the structure of the target ion channel is different, μ2-segestritoxin-Sf1a does not harm mammals. So it is safe to use it as a biological pesticide.[2]


2. BiodegradableT

he toxin is a peptide, so it must degrade over time. After degradation, the toxin will become nutrition in the soil.


3. Species-specific

According to reference, μ2-segestritoxin-Sf1a has specificity to Lepidopteran (moths) and Dipteran (flies).


4. Eco-friendly

Compare with a chemical pesticide, μ2-segestritoxin-Sf1a will not remain in soil and water so that it will not pollute the environment and won’t harm the ecosystem.


Together, using Sf1a is totally an environmentally friendly way for solving harmful insect problems by using this ion channel inhibitor as a biological pesticide.

Target insect

Figure2 Target insects


Experiment

1. Cloning


After assembling the DNA sequences from the basic parts, we recombined each PT7+B0034+Sf1a +linker+6X His-Tag gene to pSB1C3 backbones and conducted a PCR experiment to check the size of each part. The DNA sequence length of these parts is around 250-300 b.p. In this PCR experiment, the toxin product's size should be near at 550-600 b.p.
Figure 3. PT7 + RBS + Sf1a+linker+6X His-Tag
The DNA sequence length of PT7 + RBS + Sf1a+linker+6X His-Tag is around 250-300 b.p. In this PCR experiment, the product’s size should be close to 550-600 b.p.

2. Expressing


We chose E.coli Rosetta gami strain, which can form the disulfide bonds in the cytoplasm to express the protein. To verify the E.coli express the PT7 + RBS + Sf1a+linker+6X His-Tag which contains disulfide bonds, we treated the sample in two different ways. A means adding β-mercaptoethanol and sample buffer. β-mercaptoethanol can break the disulfide bonds of Sf1a and make it a linear form.

The other one adding sample buffer is the native form of PT7 + RBS + Sf1a+linker+6X His-Tag which maintains its structure. B is adding only sample buffer. The two samples are treated in boiling water for 15 mins. The SDS-PAGE shows that the native PT7 + RBS + Sf1a+linker+6X His-Tag is smaller than linear one because the disulfide bonds in PT7 + RBS + Sf1a+linker+6X His-Tag make the whole structure a globular shape.

Figure 4. PT7 + RBS + Sf1a+linker+6X His-Tag (control: Without constructed plasmid)
We can see the band of Sf1a at 5-6 kDa.
A: add β-mercaptoethanol and sample buffer
B: add sample bufferF

3. Purification


We sonicated the bacteria and purified the protein by 6X His-Tag behind the peptide using Nickel resin column. Then we ran the SDS-PAGE to verify the purification and analyze the concentration of Sf1a.
Figure 5. Protein electrophoresis of Sf1a-6X His-Tag purification
A is the sonication product.
B is the elution product of purification


4.Modeling


According to reference, the energy of Ultraviolet will break the disulfide bonds and the toxicity is also decreased. To take the parameter into consideration for our automatic system, we modeled the degradation rate of the protein and modified the program in our device.Therefore PANTIDE was be test under the Ultraviolet and model the degradation rate. the Protein Electrophoresis was showed below.
Figure 6. SDS-PAGE gel and the concentrations of UV radiolytic oxidation test to native μ-segestritoxin-Sf1a (Sf1a, 6.2 kDa). The samples are marked on the top of gel.


5. Device


We designed a device that contains a detector, a sprinkler, and an integrated hardware with users by APP through IoT talk. We use an infrared detector to detect the number of the pest and predict what time to spray the farmland. Furthermore, other detectors like temperature, humidity, lamination, pressure of carbon dioxide are also installed in our device. At the same time, the APP that displays all the information about the farmland would contact the users and spray biological pesticides automatically. This device can make farmers control the farmland remotely.


Results

Pantide-expressed E. coli Rosetta gami strain and diluted it with the three concentration.We applied the sample onto the leaf disks and put five cutworms into the separate cabinets for feeding assays. The positive control in the experiment was to apply Bacillus thuringiensis, which is the most widely-used bioinsecticide. We preserved all the result of the remained leaves sealing with the glass paper and calculated the ratio of the remained area on the leaves. The collected data were analyzed by t – test. Here are the feeding assay results.

Figure 7. Above is leaves remaining area of Negative control ( DDH2O), Positive control ( Bacillus thuringiensis bacteria ), Sf1a+linker+6XHis-Tag, Sf1a+linker+snowdrop-lectin+linker+6XHis-Tag
Figure 8. Above are leaves with of Negative control ( DDH2O), Positive control ( Bacillus thuringiensis bacteria ), Sf1a+linker+6X His-Tag


Reference

1. 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.
2. 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

Sequence and Features


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