Composite

Part:BBa_K1974011

Designed by: YU-CHUN WU   Group: iGEM16_NCTU_Formosa   (2016-10-13)
Revision as of 13:40, 30 October 2016 by PennyWu (Talk | contribs)


T7 Promoter+RBS+Hv1a+linker+6X His-Tag

Introduction:

Figure 1. PT7+RBS+Hv1a+linker+His-Tag+terminator

By ligating the IPTG induced PT7(BBa_ I712074), strong ribosome binding site (BBa_B0034), hv1a, linker, and the 6X His-Tag (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 PT7. A 6X His-Tag is added for further protein purification.

Mechanism of Hv1a:

According to the reference, Omega-hexatoxin-Hv1a has a structure called ICK (inhibitor cysteine knot). 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. [1]

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]


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.

Figure 2. Target insects


Experiment

1. Cloning


After assembling the DNA sequences from the basic parts, we recombined each PT7+B0034+toxin +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 PT7 + RBS + Hv1a+linker+6X His-Tag is around 200-250 b.p. In this PCR experiment, the product’s size should be close to 500-550 b.p.

Figure 3. PT7 + RBS + Hv1a+linker+6X His-Tag
The DNA sequence length of PT7 + RBS + Hv1a+linker+6X His-Tag is around 200-250 b.p. In this PCR experiment, the product’s size should be close to 500-550 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 + Hv1a+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 Hv1a and make it a linear form. The other one adding sample buffer is the native form of PT7 + RBS + Hv1a+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 + Hv1a+linker+6X His-Tag is smaller than linear one because the disulfide bonds in PT7 + RBS + Hv1a+linker+6X His-Tag make the whole structure a globular shape.

Figure 4. Protein electrophoresis of PT7 + RBS + Hv1a+linker+6X His-Tag (control: Without constructed plasmid)
We can see the band of Hv1a at 5-6 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 Hv1a.

Figure 5. Protein electrophoresis of Hv1a-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 ω-hexatoxin-Hv1a (Hv1a, 5.3 kDa). The samples are marked on the top of gel.


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.

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 ( DD2O ), Positive control ( Bacillus thuringiensis bacteria ), Hv1a+linker+6XHis-Tag, Hv1a+linker+snowdrop-lectin+linker+6XHis-Tag
Figure 8. Above are leaves with of Negative control ( DD2O ), Positive control ( Bacillus thuringiensis<i/> bacteria ), Hv1a+linker+6X His-Tag

Safety

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.

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. 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
2. 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.

Sequence and Features


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

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