Difference between revisions of "Part:BBa K4632003"

Revision as of 06:38, 9 October 2023

Cowpea trypsin inhibitor(CPTI)

Description

1. How does it work? CPTI can inhibite the serine protease by interacting with the active site of serine protease. Simultaneously, CPTI inhibits insect feeding by stimulating feedback signals from insect (Xuhong-lin et al.,2008)

2. Eco-friendly and Safe CPTI, a member of the Bowman-Birk protein family. Anti-insect spectrum tests show that CPTI inhibits almost all tested major agricultural pests. (Xuhong-lin et al.,2008) And, most importantly. it is Eco-friendly and Safe. (see more detail on https://2023.igem.wiki/scau-china/safety）

3. What we have done? (SCAU-China 2023) Differing from the original publication (Wei Gao et al., 2013), we did not fuse the CPTI gene with a GST purification tag for expression. Our aim is to utilize CPTI expression directly for the eradication of red fire ants, without the subsequent GST tag removal step. Furthermore, if CPTI without the GST tag can be successfully expressed, its protein molecular weight would be only 10.4 kDa.

After conducting Tricine-PAGE and Western Blot experiments, the results indicate that the CPTI gene without the GST tag does not express as expected, as described in the literature. In the next step, we have constructed the CPTI gene with the GST tag, and protein induction expression is currently underway.

Sequence and Features

Construction and Characterization

1. Verifying the Expression and Secretion Proficiency of CPTI

We ordered the OMPA-CPTI fragment from GUANGZHOU IGE BIOTECHNOLOGY LTD and inserted it into the pET30a vector.[1]

We transformed pET30a-OMPA-CPTI into E. coli BL21, induced expression with IPTG for 3 hours, and then performed centrifugation to obtain the supernatant and pellet fractions.

The supernatant was concentrated using ultrafiltration centrifuge tubes, while the pellet was subjected to disruption with lysis buffer. After disruption, we centrifuged the sample to separate the supernatant from the disrupted pellet, and the disrupted pellet was resuspended in lysis buffer.

Since 12% SDS-PAGE did not adequately display the expression results, we also attempted Tricine-PAGE and increased the SDS-PAGE separation gel concentration to 17.5%. However, upon analyzing these samples using Tricine-PAGE and Western Blot, the results revealed that the CPTI protein was not expressed in Escherichia coli (as shown in Figures 1 and 2).

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Figure 1: Tricine-PAGE results of OMPA-CPTI expression. The lanes from left to right correspond to: ①pET30a-OMPA-CPTI IPTG + concentrated supernatant ②pET30a-OMPA-CPTI IPTG - concentrated supernatant ③pET30a IPTG + concentrated supernatant ④pET30a-OMPA-CPTI IPTG + lysed whole cells ⑤pET30a-OMPA-CPTI IPTG - lysed whole cells ⑥pET30a-CPTI IPTG + lysed whole cells ⑦pET30a-CPTI IPTG - lysed whole cells ⑧pET30a IPTG + lysed whole cells ⑨pET30a IPTG - lysed whole cells ⑩Marker

Figure 2: Western Blot results of OMPA-CPTI expression. The lanes from left to right correspond to: ①pET30a-OMPA-CPTI IPTG + Concentrated supernatant ②pET30a-OMPA-CPTI IPTG - Concentrated supernatant ③pET30a IPTG + Concentrated supernatant ④pET30a-OMPA-CPTI IPTG + Lysed whole cells ⑤pET30a-OMPA-CPTI IPTG - Lysed whole cells ⑥pET30a-CPTI IPTG + Lysed whole cells ⑦pET30a-CPTI IPTG - Lysed whole cells ⑧pET30a IPTG + Lysed whole cells ⑨pET30a IPTG - Lysed whole cells ⑩Marker

In conclusion, the expression of the CPTI protein in E. coli was unsuccessful as observed in the results from both 12% SDS-PAGE, Tricine-PAGE, and 17.5% SDS-PAGE. Further investigation and optimization may be necessary to achieve successful protein expression.