Difference between revisions of "Part:BBa K1668007"

Revision as of 13:58, 16 September 2015

CDS plu1537

The part CDSplu1537 is coding sequence of toxin protein Plu1537, which is used for termite control in our project.

Plu1537 is a 14kDa insecticidal toxic protein, which had strong toxicity against termites by oral feeding according to our termites experiment. It has 30% homology with a kind of Bt toxic protein, therefore may play a similar role.

Sequence and Features

Characterization

OVERVIEW

Plu1537 is a small insecticidal toxin found in Photorhabdus luminescens, a native toxin storehouse. In our project, it is used for termite control.

We clone and standardize the gene into standard plasmid pSB1C3, and confirmed the part by PCR and sequencing. Then we combine the CDS plu1537 with arabinose inducible promoter pBad in front reporter mCherry and double terminator behind into the device plu1537 to strongly express the toxin. Termites in vivo experiments results show that Plu1537 is strongly toxic to termites.

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.

RESULTS

PLASMID CONSTRUCTION

Figure 4 digestion confirmation of tcdA1-device in pSB1A2 backbone.

5-μl samples of the single (L1) and double enzyme (L2) digestion products for plu1537-device were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. We use PstI for single digestion, XbaI and PstI for double digestion, then products were determined by AGE analysis. The DNA size standards were the DL5,000 DNA Marker (M2; TaKaRa, Cat#3428A). Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager.

It can be clearly seen the plu1537 is constructed into the pSB1C3 backbone (figure 3).

DNA SEQUNCING

We have sequenced the parts with standard primers VF2 and VR. The sequence of the 470bp part shows 100% agreement with the desired sequence.