Difference between revisions of "Part:BBa K1668008"
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[[File:TcdA1_1.jpg|200px|thumb|left|Figure 1, the 3D structure of tcdA1. Copyright 2013, Nature Publishing Group.]]
 
 
<h3> BACKGROUND </h3>
 
<h3> BACKGROUND </h3>
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[[File:TcdA1_1.jpg|200px|thumb|left|Figure 1, the 3D structure of tcdA1. Copyright 2013, Nature Publishing Group.]]
 
[[File:Prepore_and_pore.png|200px|thumb|right|Figure 2, comparison between pre-pore state and pore state of tcdA1(2, 3). Copyright 2014, Nature Publishing Group]]
 
[[File:Prepore_and_pore.png|200px|thumb|right|Figure 2, comparison between pre-pore state and pore state of tcdA1(2, 3). Copyright 2014, Nature Publishing Group]]
 
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[[File:TcdA1_2.jpg|200px|thumb|middle|Figure 3 the function of tcdA1 in toxin transportation(1). Copyright 2013, Nature Publishing Group.]]  
 
[[File:TcdA1_2.jpg|200px|thumb|middle|Figure 3 the function of tcdA1 in toxin transportation(1). Copyright 2013, Nature Publishing Group.]]  
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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.
 
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.
 
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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).  
 
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).  
 
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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).  
 
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).  

Revision as of 18:23, 14 September 2015

tcdA1-device

the part tcdA1 device is composed of arabinose inducible promoter pBad BBa_I0500, toxin protein tcdA1 coding sequnceBBa_K1668008 and composite part mCherry BBa_K1668011.

We use the device to tandem express toxic protein tcdA1 and mCherry. Toxic protein tcdA1 is a macro channel forming toxin 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
    Illegal NheI site found at 8257
    Illegal NheI site found at 8590
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1651
    Illegal BamHI site found at 1144
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 7691
    Illegal NgoMIV site found at 8351
    Illegal AgeI site found at 979
    Illegal AgeI site found at 3527
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 961


Characterization

OVERVIEW

We construct the device tcdA1 to tandem express toxic protein tcdA1 and reporter mCherry. Toxic protein tcdA1 is used to kill termites in our project.

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(1). 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. After confirmation of digestion and sequencing, we transform the plasmid into E.coli BL21(DE3) to achieve better expression level. Despite we observe that transformants have obviously turned red, we didn’t figure out the expected protein band in SDS-PAGE. Judging that the protein is considerably huge in bacteria, more improvements are needed.


BACKGROUND

Figure 1, the 3D structure of tcdA1. Copyright 2013, Nature Publishing Group.
Figure 2, comparison between pre-pore state and pore state of tcdA1(2, 3). Copyright 2014, Nature Publishing Group


Figure 3 the function of tcdA1 in toxin transportation(1). Copyright 2013, Nature Publishing Group.

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).


RESULTS