Difference between revisions of "Team:ZJU-China/Basic Part"

Latest revision as of 09:56, 18 September 2015

Part:BBa_K1668005

CDS tcdA1

The part CDS tcdA1 is the coding sequence of insecticidal protein TcdA1, which is used for termite control in our project.

tcdA1 is one of the longest genes in bacteria. And the TcdA1 toxic protein is a 285kDa pore-forming protein, belonging to tc toxic family which is widely distributed among gram-positive and gram-positive bacteria.

Sequence and Features

OVERVIEW

TcdA1, one of the biggest proteins in bacteria (285kDa), is first found in Photorhabdus luminescens. It forms channels and assists other toxins across the cell membrane. It belongs to Tc toxic protein family, which is widely distributed among different gram-negative and gram-positive bacteria.

We clone and standardize the gene into standard plasmid pSB1C3 and confirmed it by digesting and sequencing.

Background

TcdA1 is a pore-forming macro-protein, which can keep the ability to form a pore in a large pH range (from 4 to 11). To be noticed, at pH11, the pore-forming activity of TcdA1 is more than 100-fold greater than at pH6. As the midguts of most insects are alkaline, Tc toxic proteins are effective by feeding on insects, including termites.

In 2013, the structure of TcdA1 was revealed by researchers and reported in nature(1). As displayed in figure1a&b, the TcdA1 is composed of three parts: N-terminal a-helical domain(brown), the central b-sheet domain(green) and the C-terminal pore-forming domain(yellow). The protein has two states: pre-pore state and pore state. The pore-forming domain (figure 1c) sticks out to form pore, changing into pore state (figure 2).

Moreover, the TcdA1 toxin helps other toxins to enter the cell membrane. Naturally in strain TT01, TcdA1 is expressed homologously with other toxins, for example, TcdB1 and Tcc toxins. TcdA1 helps to transfer the latter into the cell to maximum the toxic effect(figure 3).

Construction

PCR

The CDStcdA1 gene was amplified by PCR by three pieces to remove three enzyme site EcoRI in its original squnce. The template is genomic DNA extracted from strain Photorhabdus luminescens TT01.

We use primer tcdA1-left L and tcdA1-left R to amplify the left side of gene., tcdA1-middle L and tcdA1-middle R to amplify the middle part and tcdA1-right L and tcdA1-right R to amplify the right side. Primers are shown below.

Seamless assembly

We used seamless assembly as our assembly method so restriction digestion and T4 ligation can be avoided. Detailed protocol and instruction for primer design can be seen in our Protocol. By this way prefix sequence, tcdA1-left, tcdA1-middle, tcdA1-right, and suffix sequence can be ligated seamlessly.

tcdA1-left F (F, 5’-3’): AATTCGCGGCCGCTTCTAGATGAACTCGCCTGTAAAAGAGA

tcdA1-left R (R, 5’-3’): AAATTCACAACAAGCGGATACATTACAC

tcdA1-middle F(F, 5’-3’): TATCCGCTTGTTGTGAATTTAATCCGG

tcdA1-middle R(R, 5’-3’): TAACATTAGCGGAAAATTCCATCACATAACCTGTTGC

tcdA1-right F(F, 5’-3’): GGAATTTTCCGCTAATGTTATGAATACCGAAGC

tcdA1-right R(R, 5’-3’): CTGCAGCGGCCGCTACTAGTATTATTATTTAATGGTGTA

Transformation and confirmation

After seamless assembly, standard plasmid pSB1C3 containing tcdA1 gene was transformed into E.coli DH5α. When single colony appeared on the LB plate, we picked out 10 colonies, respectively, as our template for bacteria solution PCR. In order to avoid the appearance of false positive clones, we used tcdA1_M1(a primer on the right part of tcdA1)/ VR as the universal primers for the CDStcdA1 is too long to amplify all the parts sequence with VF2/VR .Primers are shown below. The positive clone and its corresponding raw bacteria solution were stored and samples were sent to do DNA sequencing.

tcdA1_M1(F, 5’-3’): CTGCCAATCTATGCCACACC

VR(F, 5’-3’): ATTACCGCCTTTGAGTGAGC

Plasmid map

Fig.2 The plasmid map of BBa_K1668005 CDStcdA1

RESULTS

DNA CONSTRUCTION

Figure 4 PCR confirmation results of CDS tcdA1. We choose a 1k fragment in the middle of tcdA1 gene as template because the whole gene is much too long.

The template we use is recombinant plasmids.

It can be clearly seen the two recombinants shown in figure 4 is two positive cloning.

DNA SEQUNCING

We have sequenced the parts with standard primers VF2 and VR. The sequence of the 7.5k part shows 100% agreement with the desired sequence.

Reference

1. C. Gatsogiannis et al., NATURE 495, 520 (2013-03-20, 2013).
2. D. Meusch et al., NATURE 508, 61 (2014).

BBa_K1668006

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.

Characterization