Revision as of 12:21, 30 October 2016

T7Promoter+RBS+OAIP+linker+snowdrop-lectin+linker+6X His-Tag

Introduction:

Figure 1. P_T7+RBS+OAIP+linker+snowdrop-lectin+linker+6X His-Tag

       By ligating the IPTG induced P_T7(BBa_ I712074), strong ribosome binding site (BBa_B0034), OAIP, linker, snowdrop lectin (BBa K1974020) and the 6X His-Tag (BBa_ K1223006), we can express OAIP, the gene 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, Hadronyche versuta.
       It is under the control of the strong P_T7. Snowdrop-lectin acts as a carrier that could transport the toxin to insect’s nervous system, hemolymph and can improve the oral activity. A 6X His-Tag is added for further protein purification.
       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]

Mechanism of OAIP:

       Orally Active Insecticidal Peptide has a structure called ICK(inhibitor cysteine knot). This kind of structure contains four 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 insect voltage-gated sodium channel Site-1, making it paralyze and die eventually.

Features of OAIP:

1. Non-toxic: Orally Active Insecticidal Peptide is non-toxic to mammals. Since the structure of the target ion channel is different, Orally Active Insecticidal Peptide does not harm mammals. So it is safe to use it as a biological pesticide.^[3][4]

2. Biodegradable: Orally Active Insecticidal Peptide 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, Orally Active Insecticidal Peptide has specificity to Lepidopteran (moths), Dipteran (flies) and Orthopteran (grasshoppers).

4. Eco-friendly: Compare with chemical pesticides, 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.

Target insect:

Figure 2. Target insects

Experiment

1. Cloning :
After assembling the DNA sequences from the basic parts, we recombined each P_T7+B0034+OAIP+linker+snowdrop-lectin+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 500~600 b.p. In this PCR experiment, the toxin product's size should be near at 750-850 b.p. Proved that we successfully ligated the toxin sequence onto an ideal backbone.

Figure 3. P_T7 + RBS + OAIP+linker+Lectin+linker+6X His-Tag
The DNA sequence length of P_T7 + RBS + OAIP+linker+6X His-Tag is around 500~600 b.p. In this PCR experiment, the product’s size should be close to 750-850 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 P_T7 + RBS + OAIP+linker+Lectin+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 P_T7 + RBS +OAIP+linker+Lectin+linker+6X His-Tag and make it a linear form. The other one adding sample buffer is the native form of P_T7 + RBS + OAIPa+linker+Lectin+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_T7 + RBS+OAIP+linker+Lectin+linker+6X His-Tag is smaller than linear one because the disulfide bonds in P_T7 + RBS + OAIP+linker+Lectin+linker+6X His-Tag make the whole structure a globular shape.

Figure 4. Protein electrophoresis of P_T7 + RBS + OAIP+linker+Lectin+linker+6X His-Tag (control: Without constructed plasmid)
We can see the band of P_T7 + RBS + OAIP+linker+Lectin+linker+6X His-Tag at 17-18 kDa.
A: add β-mercaptoethanol and sample buffer
B: add sample buffer

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 P_T7 + RBS + OAIP+linker+Lectin+linker+6X His-Tag.

Figure 5. Protein electrophoresis of P_T7 + RBS + OAIP+linker+Lectin+linker+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 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.

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 6. Above is leaves remaining area of Negative control ( DDwater ), Positive control ( Bacillus thuringiensis bacteria ), Hv1a+linker+6XHis-Tag, Hv1a+linker+snowdrop-lectin+linker+6XHis-Tag

Figure 7. Above are leaves with of Negative control ( DDwater ), Positive control ( Bacillus thuringiensis bacteria ), Hv1a+linker+6X His-Tag

Safety

We also confirm the safety of applying Pantide. We sonicated the E. coliwhich 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.

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.

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. Margaret C. Hardy, Norelle L. Daly, Mehdi Mobli, Rodrigo A. V. Morales, Glenn F. King, “Isolation of an Orally Active Insecticidal Toxin from the Venom of an Australian Tarantula,” PLoS ONE, 2013, 8
4. Emily S. W. Wong1, Margaret C. Hardy1, David Wood, Timothy Bailey, Glenn F. King (2013, July). SVM-Based Prediction of Propeptide Cleavage Sites in Spider Toxins Identifies Toxin Innovation in an Australian Tarantula. PLOS ONE, 8(7), 1-11.

Sequence and Features