Difference between revisions of "Team:NCTU Formosa/Composite Part"

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Revision as of 15:12, 30 September 2016

Omega-hexatoxin-Hv1a

T7 Promoter+B0034+Hv1a+linker+6xHistag

Introduction:

By ligating the IPTG induced promoter T7 (BBa_ I712074), strong ribosome binding site (BBa_B0034), hv1a, linker, and the 6xHistag (BBa_ K1223006), we can express Hv1a, 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. Omega-hexatoxin-Hv1a is coded for the venom of a spider, Hadronyche versuta. It is under the control of the strong T7 promoter. A 6xHistag is added for further protein purification.

Mechanism of Hv1a

According to the reference, Omega-hexatoxin-Hv1a has a structure called ICK(inhibitor cysteine knot).[1] This kind of structure contains three disulfide bonds and beta-sheet. With this structure, Hv1a can resist the high temperature, acid base solution and the digest juice of insect gut. Hv1a can bind on insect voltage-gated Calcium channels (CaV1) in the central nervous system, making it paralyze and die eventually.

Features of Hv1a

1. Non-toxic: Omega-hexatoxin-Hv1a is non-toxic to mammals and Hymenoptera (bees). Since the structure of the target ion channel is different, omega-hexatoxin-Hv1a does not harm mammals and bees. So it is safe to use it as a biological pesticide.

2. Biodegradable: Omega-hexatoxin-Hv1a is a polypeptide so it must degrade over time. After degradation, the toxin will become nutrition in the soil.

3. Species-specific: According to reference, Omega-hexatoxin-Hv1a has specificity to Lepidopteran (moths), Dipteran (flies) and Orthopteran (grasshoppers).

4. Eco-friendly: Compare with chemical pesticides, Omega-hexatoxin-Hv1a will not remain in soil and water so that it will not pollute the environment and won’t harm the ecosystem.

Altogether, using Hv1a is totally an environmentally friendly way for solving harmful insect problems by using this ion channel inhibitor as a biological pesticide.

Target insect:

  • House cricket (Acheta domesticus)
  • Musca domestica
  • Amblyomma americanum
  • Heliothis virescens

    • Experiment

    1. Cloning

    After assembling the DNA sequences from the basic parts, we recombined each T7 Promoter+B0034+toxin +linker+6xHistag 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 250-500 bp. In this PCR experiment, the toxin product's size should be near at 450-700 bp.

    proved that we successfully ligated the toxin sequence onto an ideal backbone.

    2. Expressing

    E.coli(DE3) express the protein and form the disulfide in the cytoplasm. We sonicated the bacteria and purified the protein by 6xHis-tag behind the toxin using Nickel resin column.

    3. Analysis

    We do the Bradford analysis to get the protein concentration.

    Also, we do the UV test and model the degradation rate.

    We did the feeding assay on the Tabacco cutworm (Spodoptera litura) to calculate the LC50/sub>.

    放蟲抽搐、死亡之影片

    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.

    放預測降解速率的圖

    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.

    放device的真實圖

    μ-segestritoxin-Sf1a

    T7 Promoter+RBS+Sf1a+linker+6xhistag

    Introduction:

    By ligating the IPTG induced promoter T7 (BBa_ I712074), strong ribosome binding site (BBa_B0034), sf1a, linker, and the 6xHistag (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. U2-segestritoxin-Sf1a is coded for the venom of a spider, Segestria florentina. It is under the control of the strong T7 promoter. A 6xHistag is added for further protein purification.

    Mechanism of Sf1a

    U2-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.

    Features of Sf1a

    1.Non-toxic: U2-segestritoxin-Sf1a is non-toxic to mammals and bees. Since the structure of the target ion channel is different, U2-segestritoxin-Sf1a does not harm mammals and bees. So it is safe to use it as a biological pesticide.

    2.Biodegradable: The 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, U2-segestritoxin-Sf1a has specificity to Lepidopteran (moths) and Dipteran (flies). So another insect such as bees will not be killed.

    4.Eco-friendly: Compare with a chemical pesticide, U2-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 insects

  • Lucilia cuprina
  • Heliothis virescens

    • Experiment

    1. Cloning

    After assembling the DNA sequences from the basic parts, we recombined each T7 Promoter+B0034+toxin +linker+6xHistag 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 250-500 bp. In this PCR experiment, the toxin product's size should be near at 450-700 bp.

    放PCR電泳跑的圖(證明大小正確)

    proved that we successfully ligated the toxin sequence onto an ideal backbone.

    2. Expressing

    E.coli(DE3) expressed the protein and form the disulfide in the cytoplasm. We sonicated the bacteria and purify the protein by 6xHis-tag behind the toxin using Nickel resin column.

    3. Analysis

    We do the Bradford analysis to get the protein concentration.

    放SDS-PAGE圖，證明我們有表現出來

    Also, we do the UV test and model the degradation rate.

    放濃度對時間作圖

    We did the feed assay on the Tabacco cutworm (Spodoptera litura) to calculate the LC50.

    放蟲抽搐、死亡之影片

    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.

    放預測降解速率的圖

    5. Device

    We designed a device that contains detector, sprinkler, and 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 and on also install in our device. At the same time, the APP would contact the users that all the information about the farmland and spray biological pesticides automatically. This device can make farmers control the farmland remotely.

    放device的真實圖

    Orally Active Insecticidal Peptide

    T7 Promoter+RBS+Sf1a+linker+6xhistag

    Introduction:

    By ligating the IPTG induced promoter T7 (BBa_ I712074), strong ribosome binding site (BBa_B0034), OAIP, linker, and the 6xHistag (BBa_ K1223006), we are able to express OAIP, the toxin by IPTG induction.

    This year we create a revolutionary system that integrates biological pesticides, automatic detector, sprinkler, and IoT. We made a database that contains most of the spider toxins and selected the target toxins by programming. Orally Active Insecticidal Peptide is coded for the venom of a spider, Selenotypus plumipes. It is under the control of the strong T7 promoter. A 6xHistag is added for further protein purification.

    Mechanism of OAIP

    Orally Active Insecticidal Peptide has a structure called ICK (inhibitor cysteine knot). This kind of structure contains three disulfide bonds. With this structure OAIP can resist the high temperature, acid base solution and the digest juice of insect gut. OAIP can bind on the voltage-gated sodium channel in the insect’s nervous system, making it paralyze and die eventually.

    Features of OAIP

    1.Non-toxic: Orally Active Insecticidal Peptide is non-toxic to mammals and bees. Since the structure of the target ion channel is different, Orally Active Insecticidal Peptide does not harm mammals and bees. So it is safe to use it as a biological pesticide.

    2.Biodegradable: The toxin is a peptide, so it must degrade over time. After degradation, the toxin will become nutrition inside the soil.

    3.Species-specific: According to reference, Orally Active Insecticidal Peptide has specificity to Lepidopteran (moths), Coleopteran (beetles) and Isopteran (termite). So another insect such as bees will not be killed.

    4.Eco-friendly: Compare with a chemical pesticide, Orally Active Insecticidal Peptide will not remain in soil and water so that it will not pollute the environment and won’t harm the ecosystem.

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

    Experiment

    1. Cloning

    After assembling the DNA sequences from the basic parts, we recombined each T7 Promoter+B0034+toxin +linker+6xHistag 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 250-500 bp. In this PCR experiment, the toxin product's size should be near at 450-700 bp.

    放PCR電泳跑的圖(證明大小正確)

    proved that we successfully ligated the toxin sequence onto an ideal backbone.

    2. Expressing

    E.coli(DE3) express the protein and form the disulfide in the cytoplasm. We sonicated the bacteria and purified the protein by 6xHis-tag behind the toxin using Nickel resin column.

    3. Analysis

    We do the Bradford analysis to get the protein concentration.

    放SDS-PAGE圖，證明我們有表現出來

    Also, we do the UV test and model the degradation rate.

    放濃度對時間作圖

    We did the feed assay on the Tabacco cutworm (Spodoptera litura) to calculate the LC50.

    放蟲抽搐、死亡之影片

    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.

    放預測降解速率的圖

    5. Device

    We designed a device that contains detector, sprinkler, and 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 and on also install in our device. At the same time, the APP would contact the users that all the information about the farmland and spray biological pesticides automatically. This device can make farmers control the farmland remotely.

    放device的真實圖

    Omega-hexatoxin-Hv1a

    T7promoter+RBS+Hv1a+linker+snoedrop-lectin+linker+6xhistag>

    Introduction:

    By ligating the IPTG induced promoter T7 (BBa_ I712074), strong ribosome binding site (BBa_B0034), Hv1a, snowdrop-lectin, and the 6xHistag (BBa_ K1223006). Moreover, we added two kinds of the linker to connect Hv1a, snowdrop-lectin. Two linkers are different size of amino acids. We can express the toxin fused with snowdrop lectin by IPTG induction.

    This year we create a revolutionary system that integrates biological pesticides, automatic detector, sprinkler, and IoT. We made a database that contains most of the spider toxins and selected the target toxins by programming. Omega-hexatoxin-Hv1a is coded for the venom of a spider, Hadronyche versuta.

    It is under the control of the strong T7 promoter. Snowdrop-lectin acts as a carrier that could transport the toxin to insect’s nervous system, hemolymph and can improve the oral activity. A 6xHistag is added for further protein purification.

    Mechanism of Hv1a

    According to reference, snowdrop-lectin is resistant to high temperature and would not be degraded by digestive juice. The species-specificity is based on the toxin, and the snowdrop lectin is the role of the carrier.

    According to reference, Omega-hexatoxin-Hv1a has a structure called ICK(inhibitor cysteine knot).[1] This kind of structure contains three disulfide bonds and beta-sheet. With this structure, Hv1a can resist the high temperature, acid-base solution and the digest juice of insect gut. Hv1a can bind on insect voltage-gated Calcium channels (CaV1) in the central nervous system, making it paralyze and die eventually.

    Features of Hv1a

    1.Non-toxic: Omega-hexatoxin-Hv1a is non-toxic to mammals and Hymenoptera (bees). Since the structure of the target ion channel is different, omega-hexatoxin-Hv1a does not harm mammals and bees. So it is safe to use it as a biological pesticide.

    2.Biodegradable: Omega-hexatoxin-Hv1a is a polypeptide so it must degrade over time. After degradation, the toxin will become nutrition in the soil.

    3.Species-specific: According to reference, Omega-hexatoxin-Hv1a has specificity to Lepidopteran (moths), Dipteran (flies) and Orthopteran (grasshoppers).

    4.Eco-friendly: Compare with chemical pesticides, Omega-hexatoxin-Hv1a will not remain in soil and water so that it will not pollute the environment and won’t harm the ecosystem.

    Together, using Hv1a is totally an environmentally friendly way for solving harmful insect problems by using this ion channel inhibitor as a biological pesticide.

    Target insect:

  • House cricket (Acheta domesticus)
  • Musca domestica
  • Amblyomma americanum
  • Heliothis virescens

    • Experiment

    1. Cloning

    After assembling the DNA sequences from the basic parts, we recombined each T7 Promoter+B0034+toxin +linker+6xHistag 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 250-500 bp. In this PCR experiment, the toxin product's size should be near at 450-700 bp.

    放PCR電泳跑的圖(證明大小正確)

    proved that we successfully ligated the toxin sequence onto an ideal backbone.

    2. Expressing

    E.coli(DE3) express the protein and form the disulfide in the cytoplasm. We sonicated the bacteria and purified the protein by 6xHis-tag behind the toxin using Nickel resin column.

    3. Analysis

    We do the Bradford analysis to get the protein concentration.

    放SDS-PAGE圖，證明我們有表現出來

    Also, we do the UV test and model the degradation rate.

    放濃度對時間作圖

    We did the feed assay on the Tabacco cutworm (Spodoptera litura) to calculate the LC50.

    放蟲抽搐、死亡之影片

    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.

    放預測降解速率的圖

    5. Device

    We designed a device that contains detector, sprinkler, and 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 and on also install in our device. At the same time, the APP would contact the users that all the information about the farmland and spray biological pesticides automatically. This device can make farmers control the farmland remotely.

    放device的真實圖