Part:BBa_K1668010

plu1537-device

the part plu1537 device is composed of arabinose inducible promoter pBad 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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1205
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 1144
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 979
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 961
  

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.

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).

PLASMID SEQUNCING

We have sequenced the parts with standard primers VF2 and VR. The sequence of the 2.5k part shows 100% agreement with the desired sequence.

TOXIN EXPRESSION

BACTERIA CULTURE

Figure 4 expression of reporter mCherry in LB solid medium with arabinose and chloromycetin.

Figure 5 expression of reporter mCherry in LB liqiud medium with arabinose and chloromycetin.

The solid or liquid culture medium is LB culture with 34ug/ml chloromycetin and 80mM arabinose.
Both the antibiotics and arabinose are added after the culture cools down to 60℃. 2%(w/v) of agar is added in solid medium.
It can be clearly seen that the recombinant turned red, indicating the expression of reporter mCherry(figure 4, 5). As mCherry is located behind target gene and shares a promoter with target gene, the target gene may be expressed to a great extent.

SDS-PAGE

Figure 6 SDS-PAGE results of four devices we constructed.

5ul sample is loaded in a 10% SDS-PAGE separation gel. We use 250kDa marker Precision Plus Protein™ Dual Color Standards #161-0374. Parameters can be seen in protocols.

According to the result of SDS-PAGE (figure 6), target protein (14kDa) is strongly expressed (line 3) compared with the negative control, native BL21 (DE3) strain without engineering (line 1).

TERMITES in vivo EXPERIMENTS

内容暂缺