Part:BBa_K1974011

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

Introduction:

Figure 1. T₇ promoter+RBS+Hv1a+linker+His-tag+terminator

      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). 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.

Figure 2. Hv1a 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 PT₇ + RBS + Hv1a+linker+6X His-Tag is around 200-300 b.p. In this PCR experiment, the product’s size should be close to 500-600 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 200-300 b.p. In this PCR experiment, the product’s size should be close to 500-600 b.p.

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.

Figure 4. Protein Electrophoresis of PT₇ + RBS + Hv1a+linker+6X His-Tag (control: Without constructed plasmid)
Hv1a is an around 50 amino acids, so we can see the band of Hv1as at 5-6 kDa. The linear Hv1a contains β-mercaptoethanol, which breaks the disulfide bonds of Hv1a. The native Hv1a doesn’t have β-mercaptoethanol, so it can maintain its structure. Below are different method of treating sample of Hv1a+linker+6X His-Tag abbreviation:
Linear: add β-mercaptoethanol and sample buffer Native: only add sample buffer

Figure 5. Protein Electrophoresis of Hv1a+linker+6X His-Tag purification Hv1a is an around 50 amino acids, so we can see the band of Hv1as at 5-6 kDa. Below are fragment of serial purification of Hv1a+linker+6X His-Tag abbreviation: Before: Before purification PT: Pass through W1~W3: Washing Buffer E1~E10: Elution Buffer N:NaCl

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.

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.

Figure 8. Below are leaves with of Negative control ( DDwater ), Positive control ( Bacillus thuringiensis bacteria ), Hv1a+linker+6X His-Tag

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
  INCOMPATIBLE WITH RFC[25]

  Unknown
• 1000
  COMPATIBLE WITH RFC[1000]