Difference between revisions of "Part:BBa K1668010"

Revision as of 13:42, 15 September 2015

_NOTOC__ plu1537-device the part plu1537 device is composed of arabinose inducible promoter pBad[https://parts.igem.org/Part:

BBa_I0500], toxin protein plu1537 coding sequence BBa_K1668007 and

composite part mCherry BBa_K1668011.

 We use the device to tandem express toxic protein Plu1537 and mCherry in DH5α BL 21(DE3). 

The toxin protein is used for termite control in our project and mCherry is a reporter.

Sequence and Features No part name specified with partinfo tag.

Characterization

OVERVIEW

We construct the device plu1537 to tandem express insecticidal toxic protein Plu1537 and reporter

mCherry. The 14kDa insecticidal toxic protein Plu1537 is used to kill termites in our project.

The exact mechanism of insecticidal toxin protein Plu1537 remains unclear. In a 2009 research, scientists

first expressed the plu1537 and purified the Plu1537 with GST tag. As a result, Plu1537 had insecticidal

activity against two moth species (Galleria& Spodoptera) larvae via hemocoel injection instead of oral

feeding.

We clone and standardize the gene into standard plasmid pSB1C3. After confirmation of digestion and

sequencing, we transform the plasmid into E.coli BL21(DE3) to achieve better expression level.

We observe that transformants have obviously turned red and figure out the expected protein band in SDS-PAGE.

According to in vivo experiments on termites, the toxin effect of Plu1537 is far more ideal than our expected

by oral feeding.

In conclusion, we have successfully cloned and expressed the Plu1537 toxic protein in DH5α BL 21(DE3)

. Toxin effects of oral feedings on termites are far better than that described in research on moth

larvae.

BACKGROUND

Figure 1, the 3D structure of cry34Ab1. Copyright 2014, Worldwide Protein Data.

Figure 2, comparison between cry34Ab1 and cry35Ab1(2). Copyright 2014, Public Library of Science.

In 2009 research Expression and activity of a probable toxin from Photorhabdus Luminescens, toxin

protein pit, which is 94% homologous with plu1537, is expressed in DH5α BL21(DE3). Engineered strain

BL21 was both orally fed and injected in hemocoel to two kind of moth(Galleria mellonella & Spodoptera

litura)(1). As a result, hemocoel injection is more effective than oral feeding. However, our

experiment showed that oral feeding is also effectively.

Plu1537 shares 30% amino acid sequence similarity with a 13.6 kDa insecticidal crystal protein cry34Ab1 in

Bacillus thuringiensis (figure 1), which belongs to bt toxin family.

Bt family may be the most famous insecticidal toxin up to now. It’s one of biological toxins first used for

pest control. After years of study, bt family is becoming bigger and bigger. Basically, most proteins in bt

family form pores in cell membrane to kill a cell, including cry34Ab1(2).

In 2014, the structure of cry34Ab1 was revealed and reported in PLOS ONE. As displayed in figure 1, the

structure of cry34Ab1 is simpler, compared with other two toxins we used. However, cry34Ab1 (figure 2A) can

only function with the assistant of cry35Ab1 (figure 2B)(2), which differentiate cry34 Ab1 from

Plu1537, which is toxic without any other assistant.