Difference between revisions of "Part:BBa K1668006"

Revision as of 12:41, 16 September 2015

CDS plu0840

The part CDSplu0840 is coding sequence of toxin protein Plu0840, which is used for termite control in our project.

Plu0840 is a 72kDa insecticidal toxic protein, which had weak oral toxicity against two kinds of moth according to a 2007 research and showed weak toxicity to termites by oral feeding.

Sequence and Features

Characterization

OVERVIEW

Plu0840 is a 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 plu0840 with arabinose inducible promoter pBad in front reporter mCherry and double terminator behind into the device plu0840 to strongly express the toxin.

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 DH5α BL21(DE3). Engineered strain BL21 was both orally fed and injected in hemocoel to two kind of moth (S. litura and S.exigua)(1).

According to the result, on the one hand, oral feeding effectively inhibits the growth of larva while 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 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 4 digestion confirmation of tcdA1-device in pSB1A2 backbone.

5μl samples of the double enzyme digestion products for tcdA1-device were loaded onto a 1% BioRad Ready Agarose Mini Gel, then subjected to AGE. See (protocol) for AGE parameters. Sizes of the XbaI and PstI–cleaved assemblies were determined by AGE analysis. The DNA size standards were the DL5,000 DNA Marker (M2; TaKaRa, Cat#3428A) and 1kb DNA Ladder (Dye Plus)(M2; TaKaRa, Cat#3426A). Bands were visualized with a Shanghai Peiqing JS-380A Fluorescence Imager.

First we construct the tcdA1 device in pSB1A2. Our target fragments can be clearly seen in the right position (figure 4). As the fragment is a little big(7.2k), the efficiency is low when we change the backbone to pSB1C3 and the unwanted fragment is hard to explain(figure 5).

PLASMID SEQUNCING

We have sequenced the parts with standard primers VF2 and VR. The sequence of the 1.8k part shows 100% agreement with the desired sequence.