Part:BBa_K1668009

plu0840-device
the part plu0840 device is composed of arabinose inducible promoter pBad BBa_I0500, toxin protein plu0840 coding sequence BBa_K1668006 and composite part mCherry BBa_K1668011.

We use the device to tandemly express toxic protein Plu0840 and mCherry inE.coli BL21(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 plu0840 to tandemly express insecticidal toxic protein Plu0840 and reporter mCherry. The 72kDa insecticidal toxic protein Plu0840 is used to kill termites in our project.

The exact mechanism of insecticidal toxin protein Plu0840 remains to be revealed. In a 2007 research, scientists first expressed a GST-plu0840 fusion protein in E.coli BL21(DE3) with pGEX-4T-1 vector. The results showed that Plu0840 had weak oral toxicity against two kinds of moth and inhibited their growth (S. litura and S. exigua)

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 Plu0840 is comparatively weaker than Plu1537, which is consistent with the research before.

In conclusion, we have successfully cloned and expressed the Plu0840 toxic protein in E.coli BL21(DE3). The Plu0840 is weakly toxic to termites.

BACKGROUND

Figure 1 The 3D structure of Plu0840. Copyright 2014, Worldwide Protein Data.

In 2009 research Cloning and expression analysis of a predicted toxin gene from Photorhabdus sp. HB78, plu0840 fused with GST is expressed in E.coli BL21(DE3). Engineered strain BL21 was both orally fed and injected in hemocoel to two kind of moths (S. litura and S.exigua)(1).

According to the result, on the one hand, oral feeding effectively inhibits the growth of larva while Plu0840 has only weak oral toxic effect. On the other hand, hemocoel injection showed negative results.

The research mentions that Plu0840 (Figure 1) shares 55% sequence identity with an enterotoxin Ast from aeromonas hydrophila. Aaeromonas hydrophila, which is connected with gastroenteritis, may lead to altered fluid secretion in mouse. According to a 2002 research, enterotoxin Ast makes weak contributions to fluid secretion compared with two proteins produced by other genes (2) However, judging that TT01 is nontoxic to animals at all, we think Plu0840 may share little similarity with Ast.

RESULTS

PLASMID CONSTRUCTION

Figure 2 Digestion confirmation of device plu0840 in pSB1C3 backbone.

5-μl samples of the single (L1) and double enzyme (L2) digestion products for plu0840-device were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See protocol http://2015.igem.org/Team:ZJU-China/Project/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 plu0840 is constructed into the pSB1C3 backbone (Figure 2).

PLASMID SEQUNCING

We have sequenced the parts with standard primers VF2 and VR. The sequence of the 3.9k part shows 100% agreement with the desired sequence.

TOXIN EXPRESSION

BACTERIA CULTURE

Figure 3 Expression of reporter mCherry in LB solid medium with arabinose and chloromycetin.

Figure 4 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 5 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 5), target protein Plu0840 (72kDa) is strongly expressed (line 3) compared with the negative control, native BL21(DE3) strain without engineering (line 1).

TERMITES in vivo EXPERIMENTS

Figure 6 Toxin effect of Plu0840 on termites.

We fed the same amount of termites separately with Plu0840-expressing BL21, Plu0840-expressing BL21 embedded with CNCs and control group: mCherry-expressing BL21</i>. For more details please go to our Protocol http://2015.igem.org/Team:ZJU-China/Project/Protocol

In six days, three termites died in Plu0840-expressing group and two in CNCs group. We conclude that Plu0840 may have weak oral toxicity on termites, which also corresponds with results of related research. However, more experimental proof is needed to further confirm the toxicity of Plu0840.

REFERENCE

1. M. Li et al., MOL BIOL REP 36, 785 (2009).
2. J. Sha, E. V. Kozlova, A. K. Chopra, INFECT IMMUN 70, 1924 (2002).
3. Plu0840 (uniprot): http://www.uniprot.org/uniprot/Q7N895.
4. 3D structure of Plu0840: http://www.proteinmodelportal.org/?pid=modelDetail&provider=MODBASE&template=1uasA&pmpuid=1000725544546&range_from=1&range_to=639&ref_ac=Q7N895&mapped_ac=Q7N895&zid=async.