Coding

Part:BBa_K4632003

Designed by: Haolong Lai   Group: iGEM23_SCAU-China   (2023-10-08)
Revision as of 20:42, 8 October 2023 by LHL scau (Talk | contribs)


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


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]

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 (plasmid map shown as follow).


<figure align="center"> <img alt="parts1" src="part-2-1.png" width="200" title="Construction of pET30a-OmpA-CPTI"

<figcaption> Figure 1: Construction of pET30a-CPTI </figcaption>

</figure>



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).

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.





[edit]
Categories
Parameters
None