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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 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.		<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 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.
−	[[File:Hv1a Lectin GS linker NCTU Formosa.png|200px|thumb|center|'''Figure 3.'''The DNA sequence length of PT7+Hv1a+linker+lectin+linker+6X His-Tag is around 500-600 b.p. In this PCR experiment, the product’s size should be close to 800-900 b.p.]]	+	[[File:Hv1a Lectin GS linker NCTU Formosa.png|200px|thumb|center|'''Figure 3.'''The DNA sequence length of PT7+Hv1a+GS linker+lectin+linker+6X His-Tag is around 500-600 b.p. In this PCR experiment, the product’s size should be close to 800-900 b.p.]]
	<p style="padding:1px;font-size:16px"><b>2. Expressing</b></p><br><i>E. coli</i>(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.		<p style="padding:1px;font-size:16px"><b>2. Expressing</b></p><br><i>E. coli</i>(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.
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	<p style="padding:1px;font-size:16px"><b>3. Purification</b></p><br>Protein electrophoresis of Hv1a-linker-Lectin-linker-6X His-Tag purification. A is the sonication product. B is the elution product of purification.		<p style="padding:1px;font-size:16px"><b>3. Purification</b></p><br>Protein electrophoresis of Hv1a-linker-Lectin-linker-6X His-Tag purification. A is the sonication product. B is the elution product of purification.
−	[[File:HGL-p.png|300px|thumb|center|'''Figure 4.'''Protein electrophoresis of P<sub>T7</sub> + RBS + Hv1a+linker+Lectin+linker+6X His-Tag purification.	+	[[File:HGL-p.png|300px|thumb|center|'''Figure 4.'''Protein electrophoresis of P<sub>T7</sub> + RBS + Hv1a+GS linker+Lectin+linker+6X His-Tag purification.
	<br>A is the sonication product. B is the elution product of purification.]]		<br>A is the sonication product. B is the elution product of purification.]]
	<!--Also, we do the UV test and model the degradation rate.-->		<!--Also, we do the UV test and model the degradation rate.-->
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−	[[File:NCTU FORMOSA OL-6.png|400px|thumb|center|'''Figure 5.'''Below are leaves with of Negative control ( DDH<sub>2</sub>O ), Positive control ( <i>Bacillus thuringiensis</i> bacteria ), PT7-Hv1a-linker-Lectin-linker-6X His-Tag]]	+	[[File:NCTU FORMOSA OL-6.png|400px|thumb|center|'''Figure 5.'''Below are leaves with of Negative control ( DDH<sub>2</sub>O ), Positive control ( <i>Bacillus thuringiensis</i> bacteria ), PT7-Hv1a-GS linker-Lectin-linker-6X His-Tag]]
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	<!---放ＤＥＶＩＣＥ真時圖------>		<!---放ＤＥＶＩＣＥ真時圖------>
−	<h1>'''Safety'''</h1>
−	<p style="padding:1px;">We also confirm the safety of applying Pantide. We sonicated the <i>E. coli</i>which expressed Hv1a-lectin and treat it in boiling water about 4 hours and collect the product of  interval for 0.5 hour. Then we cultured the products  after boiling for about a week and found that there's no bacteria growing in the LB plate. Through this experiment, we could make sure that the product is proved to be safe. Here we see the result.</p>
−	[[File: Safety_plate.jpeg|400px|thumb|center|'''Figure 9.''' The LB plate is divided into 9 parts. Each of parts is different time interval boiling in water. The control group is only LB solution. The other 8 ones are boiling for 1, 1.5, 2, 2.5. 3, 3.5, 4 hours separately. ]]	+	<h1>'''Safety'''</h1>
		+	<p style="padding:1px;">
		+	We do the safety part which is the same as the Hv1a-lectin. See more in
		+	<html><a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K1974021">BBa_K1974021</a></html>.

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Latest revision as of 23:20, 30 October 2016

T7 Promoter+RBS+Hv1a+GS linker+snowdrop-lectin+linker+6X His-Tag

Introduction:

By ligating the IPTG induced promoter T7 (BBa_ I712074), strong ribosome binding site (BBa_B0034), hv1a, linker,,snowdrop lectin( BBa_K1974020), 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, 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.

This year, we have designed fusion proteins of spider toxin in conjunction with snowdrop lectin by the three-alanine linker. Through the reference searching, we found that the previous research all used the three-alanine linker. At first, we planned to use the Flexible Peptide GS Linker(BBa_K416001)from 2010 NYU iGEM team. However, we found no result in the previous history using this linker. In the experiment, the fusion protein with the elongated GS linker did show the enhanced repellent effect. Observed from the phenomenon, we did prove the enhanced function of the fusion proteins, so we speculate that the fusion protein has a better secondary structure than the original ones. As a result, we have submitted a basic part for the improved elongated GS linker.

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.^[1][2]

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.^[3][4]

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:

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. Purification

Protein electrophoresis of Hv1a-linker-Lectin-linker-6X His-Tag purification. A is the sonication product. B is the elution product of purification.

Also, we use tabacco cutworm to test it's effect. Accoding to the Histogram show below, the dose response proof that Hv1a is really work. The negative control are water and the positive control are commercially available pesticides. Leaves in the table are direct results.

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 modify the program in our device.

5. Device

We designed a device that contains detector, sprinkler, and integrated hardware with users by APP through IoT talk. We use 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.

Safety

We do the safety part which is the same as the Hv1a-lectin. See more in BBa_K1974021.

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. Wang, X.H.; Connor, M.; Wilson, D.C.; Wilson, H.I.; Nicholson, G.M.; Smith, R.; Shaw, D.; Mackay, J.P.; Alewood, P.F.; Christie, M.J.; King, G.F. “Discovery and structure of a potent and highly specific blocker of insect calcium channels,” J. Biol. Chem. 2001, 276, 40306–40312 5. Erich Yukio, Tempel Nakasu (2014, July). Effects of ω-ACTX-Hv1a/GNA, a novel protein biopesticide targeting voltage-gated calcium ion channels, on target and non-target arthropod species. Retrieved from http://hdl.handle.net/10443/2643.

Sequence and Features