Part:BBa_K1974012

T7Promoter+RBS+Sf1a+linker+6X His-Tag

Introduction:

Figure 1.P_T7+RBS+Sf1a+linker+His-Tag+terminator

      By ligating the IPTG induced P_T7(BBa_ I712074), strong ribosome binding site (BBa_B0034), sf1a, linker, and the 6X His-Tag (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 P_T7. A 6X His-Tag 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 insect:

Figure2Sf1a target insect

Experiment

1. Cloning :
After assembling the DNA sequences from the basic parts, we recombined each P_T7+B0034+Sf1a +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 250-300 b.p. In this PCR experiment, the toxin product's size should be near at 550-600 b.p.

Figure 3.P_T7 + RBS + Sf1a+linker+6X His-Tag
The DNA sequence length of P_T7 + RBS + Sf1a+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:
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 + Sf1a+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 Sf1a and make it a linear form. The other one adding sample buffer is the native form of P_T7 + RBS + Sf1a+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 + Sf1a+linker+6X His-Tag is smaller than linear one because the disulfide bonds in P_T7 + RBS + Sf1a+linker+6X His-Tag make the whole structure a globular shape.

Figure 4.P_T7 + RBS + Sf1a+linker+6X His-Tag (control: Without constructed plasmid)
We can see the band of Sf1a 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 Sf1a.

Figure 5.Protein electrophoresis of Sf1a-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 μ-segestritoxin-Sf1a (Sf1a, 6.2 kDa). The samples are marked on the top of gel.

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.

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.Below are leaves with of Negative control ( DDwater ), Positive control ( Bacillus thuringiensis bacteria ), Sf1a+linker+His-Tag, Sf1a+linker+snowdrop-lectin+linker+His-Tag

Figure 8.Below are leaves with of Negative control ( DDwater ), Positive control ( Bacillus thuringiensis bacteria ), Sf1a+linker+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
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]