Difference between revisions of "Team:NCTU Formosa/Proof"

Cloning Result

Gene Constructions of Pantide—PCR

To ensure the genes are cloned into E. coli BL21 Rosetta-gami strain, we did electrophoresis of PCR products for checking insert gene’s length after amplification with PCR. Each of the BioBrick construction includes the conservative promoter, RBS, linker, and His-tag. Here are the theoretical length of seven BioBricks we construct.(Table 1)

After amplification with PCR, the PCE products of Hv1a, Sf1a, and OAIP have a length of around 500bp； and Hv1a-Lectin, Sf1a-Lectin, and OAIP-Lectin has a length of 750bp~1000bp； and Hv1a-Lectin with GS linker has a length of 893bp. (Figure 1a, 1b, 1c, 1d, 1e, 1f, 1g)

Figure 1. Polymerase chain reaction (PCR) amplification analysis of electrophoresis of seven PCR products with Marker labeled length on the left hand (a)Hv1a (BBa_K1974011, 495 base pairs). (b)Sf1a (BBa_K1974012, 522 base pairs). (c)OAIP (BBa_K1974013, 489 base pairs). (d)Hv1a-Lectin (BBa_K1974021, 819 base pairs). (e)Sf1a-Lectin (BBa_K1974022, 846 base pairs). (f)OAIP-Lectin (BBa_K1974023, 813 base pairs). (g)Hv1a-Lectin with GS linker(BBa_K1974033, 883 base pairs).

Expression Result

Expression of Pantide

Our goal is to express Pantide successfully. We used E. coli BL21 rosetta-gami strain to cultivate genes. Afterward, we sonicated E. coli and then ran SDS-PAGE to make sure that Pantide fell into the correct ranges. There are seven kinds of BioBricks, and the mass of them are showed below.(Table 2)

The gel of SDS-PAGE result is correct, and here is our result.(Figure 2a, 2b, 2c)

Figure 2. SDS-PAGE result of the six Pantide-expressed sonicated products compared with the unexpressed sonicated one as a control and with Marker labeled length on the left hand. A: add β-mercaptoethanol and sample buffer, B: add sample buffer. (a)Hv1a (5.3 kDa) (b)Sf1a (6.2 kDa) (c)OAIP (5.3 kDa) (d)Hv1a-Lectin (17.1 kDa) (e)Sf1a-Lectin (18.1 kDa) (f)OAIP-Lectin (17.2 kDa)

Purification of Pantide

After sonicated E. coli, we further purified Pantide to check whether it is well-expressed. The purified products’ length can be seen in the table below.(Table 3)

The gel of SDS-PAGE result of Hv1a, Sf1a, OAIP have a length of 5kDa~7 kDa；and Hv1a-Lectin, Sf1a-Lectin, OAIP-Lectin, and Hv1a-GS linker-Lectin have a length of 17kDa~19kDa.(Figure 3a, 3b, 3c, 3d, 3e, 3f, 3g) As a result, the constructions of BioBrick were correct.

Figure 3. SDS-PAGE result of the seven purified Pantide products compared with the unpurified one, and with Marker labeled length on the left hand. A is the sonication product. B is the elution product of purification. (a) Hv1a (5.3 kDa) (b) Sf1a (6.2 kDa) (c) OAIP (5.3 kDa) (d) Hv1a-Lectin (17.1 kDa) (e) Sf1a-Lectin (18.1 kDa) (f) OAIP-Lectin (17.2 kDa) (g) Hv1a-GS linker-Lectin (18.2 kDa)

Feeding Assays

To understand the insect test in an easy way, the testee selection, and experiment design would be briefly described. The results consist of three parts-feeding assay pre-test, the fusion protein improvement test, and preference test.
**Note: The word “Pantide” in the following paragraph refers to the collection of all the toxin design in our project.

• Testee Selection

  In the test of Pantide toxicity, we chose tobacco cutworm as the testee for the insect appetite tests because all the Pantide targets lepidopterans. We hope that we can observe the difference of the three toxins in vivo. Tobacco cutworms, which are one of the major pests in vegetable farms, impact on Crucifers in farmland around the world. In the serious pest damage, the density of tobacco cutworms is approximately up to two hundred million per hectare. Therefore, we used five tobacco cutworms to mimic the much more severe situation in our insect experiment.

• Experiment Design

  All the experiments were to check the functions of Pantide for leaf protection, so the observation of the results would focus on the remained area of the leaf disks.
         In the following experiments, we used E. coli BL21 Rosetta-Gami strain to produce Pantide. We aimed to evaluate the remained leaf area applying Pantide. In this insect test, Pantide was diluted into three concentrations to observe the trends of dose response and to confirm the quality of PANTIDE. All the following experiments have three dilution ratios including 1/125 x, 1/25x and 1/5x. Three repeated tests were done in each experiment. See the procedure of insect experiment on Protocols.

• Hypothesis

  According to the mechanism of Pantide, Pantide is supposed to perform its toxicity in the nervous system of the larvae. We might see larvae paralyze and finally die.

Insect Test Result

Feeding Assay Pre-test

To know the qualitative toxicity effect of Pantide, we prepared the samples with the sonicated LB solution lysate containing Pantide-expressed E. coli Rosetta-Gami strain and diluted it with the three concentration. This experiment is the pre-test that shows us whether the amount of Pantide is sufficient enough to perform the toxicity against the larvae. 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 on the leaf disks, which is nowadays the most widely-used bioinsecticide, and the negative control group in the test was DD water. We preserved all the result of the remained leaves sealing with the glass paper and calculated the percentage of the remained area on the leaves. The collected data were analyzed by t–test and calculated with the p-value. The p-value is a function that measures how extreme the observation is. The widespread use of “statistical significance” (interpreted as “p ≤ 0.05”) is a license for making a claim of a scientific finding that leads to considerable distortion of the scientific process. In the picture, it shows as a star. The more the stars are, the more significant difference is. Here are the feeding assay results.

Feeding Assay Pre-test of Hv1a and Hv1a-lectin

Figur1. Remained leaf disks in the pre-test with the Hv1a and Hv1a-lectin.

Figure2. The dose response analysis of Hv1a/Hv1a-lectin.

Figure3. The T-test analysis in different dose of Hv1a/Hv1a-lectin.

Figure1 shows the pictures of the remained leaf disks after twelve hours of feeding assays. After we had done the feeding assays on tobacco cutworms with the three dilution ratios, we measured the area with the software imageJ. Figure2 shows the percentage of the remained area on the leaf disks. The higher the bar is, the larger the remained leaves area is. Figure3 shows the p-value with the star attached to indicate P < 0.05 and two stars to indicate P < 0.01. The p-value of the T-test can be used to determine if two sets of data are significantly different from each other. The smaller the p-value is, the more significant the differences are between the two groups. The observed phenomenon can be analyzed below.

• With the dilution of Hv1a/Hv1a-lectin, the remained leaves area decreased, which indicates the dose response. The dose response is shown in figure2.
• Compared Hv1a with Hv1a-lectin, the remained leaves area in the Hv1a-lectin is higher than that of Hv1a, which means the repellent efficiency of Hv1a-lectin is higher than that of Hv1a. It shows in figure2.
• To use the p-value as an indicator of statistical significance, we compared Hv1a/Hv1a-lectin with the negative control; it shows that the remained leaf disk area of Hv1a and Hv1a-lectin are significantly different from negative control, shown in figure3.

To sum up, the Hv1a and Hv1a-lectin work well in vivo. The effect can be roughly ranked as:
Hv1a-lectin > Hv1a > E. coli > Negative control

We got the similar result in the Sf1a/Sf1a-lectin and OAIP/OAIP-lectin.

Feeding Assay Pre-test of Sf1a and Sf1a-lectin



Figure4. Remained leaf disks in the pre-test with the Sf1a and Sf1a-lectin.



Figure5. The dose response analysis of Sf1a/Sf1a-lectin.



Figure6. The T-test analysis in different dose of Sf1a/Sf1a-lectin.

Feeding Assay Pre-test of OAIP and OAIP-lectin



Figure7. Remained leaf disks in the pre-test with the OAIP and OAIP-lectin.



Figure8. The dose response analysis of OAIP/OAIP-lectin.



Figure9. The T-test analysis in different dose of OAIP/OAIP-lectin.

The Fusion Protein Improvement Test

In this experiment, we compared the toxicity effect of the three toxin designs including Hv1a/Hv1a-lectin/Hv1a-lectin with GS linker to test the toxicity effect. (For more about the improved Pantide with GS linker, see the link)



Figure10. Remained leaves in the GS-linker improvement test with the Hv1a, Hv1a-lectin, and Hv1a-lectin GS-linker improved.



Figure11. The T-test analysis in different dose of GS-linker improvement test.

To know how the length of the linker affect the function of the fusion protein, we constructed Hv1a-lectin with the original three-alanine linker and a longer GS linker. Besides, there is also a Hv1a without the linker between the repellent peptide and the lectin served as a comparative sample. Figure10 shows the pictures of the remained leaf disks after twelve hours of feeding assays. After we had done the feeding assays on tobacco cutworms with the three dilution ratio, we measured the area with the software imageJ. Figure11 shows the ratio of the remained area on the leaf disks. The higher the bar is, the larger the remained leaves area is. The observed phenomenon can be analyzed below.

• We purified Pantide into three quantitative concentration with 0.4μM, 2μM, and 10 μM. The remained leaves area decreased as the concentration of PANTIDE decreased, which shows the dose response of Pantide.
• In comparison with the two other design, we could find out that the remained leaves area with improved Hv1a-lectin were all more than that of the original Hv1a-lectin and Hv1a. The result shows that by enhancing the length of the linker, the fusion protein works performs its toxicity better.

Preference Test

In the two-hour test, we can see that most of the tobacco cutworms move to eat the negative control group, which proves the repellent effect of Pantide.

Conclusion

After the three experiments, we have three conclusions.

• Pantide performs its toxicity as a repellent in vivo. It is a noticeable finding that goes beyond our original hypothesis. The mechanism of the repellent effect is so far unknown.
• The toxicity efficacy of Pantide is equal to Bacillus thuringiensis.
• With the elongation of the linker, the improved fusion protein has the best repellent effect.
• The tobacco cutworms have a preference on the leave disks without applying Pantide.

Although Pantide does not act as a biological pesticide as our hypothesis had mentioned, we may conclude that it is a biological insect repellent, which corresponds to our ideology of lowering the artificial impacts on the environment and simultaneously achieve effective pest control.