Difference between revisions of "Part:BBa K1668005"
 
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<partinfo>BBa_K166805 short</partinfo>
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<partinfo>BBa_K1668005 short</partinfo>
  
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The part CDS <i>tcdA1</i> is the coding sequence of insecticidal protein tcdA1, which is used for termite control in our project.
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The part CDS <i>tcdA1</i> is the coding sequence of insecticidal protein TcdA1, which is used for termite control in our project.
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<i> tcdA1</i> 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.
 
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<i> tcdA1</i> 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.
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<h2>'''Characterization'''</h2>
 
<h2>'''Characterization'''</h2>
 
<h3> OVERVIEW </h3>
 
<h3> OVERVIEW </h3>
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tcdA1, one of the biggest proteins in bacteria (285kDa), is first found in <i>Photorhabdus luminescens</i>. It forms channels and assists other toxins across the cell membrane(<i>1</i>). It belongs to tc toxic protein family, which is widely distributed among different gram-negative and gram-positive bacteria.  
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TcdA1, one of the biggest proteins in bacteria (285kDa), is first found in <i>Photorhabdus luminescens</i>.
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<br>
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It forms channels and assists other toxins across the cell membrane(<i>1</i>).  
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<br>
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It belongs to Tc toxic protein family, which is widely distributed among different gram-negative and gram-positive bacteria.  
 
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<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.]]
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[[File:ZJU-CHINA_tcdA1_structure.jpg|200px|thumb|left|Figure 1 The 3D structure of TcdA1. Copyright 2013, Nature Publishing Group.]]
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[[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:ZJU-CHINA_A1_prepore_and_pore.png|200px|thumb|right|Figure 2 Comparison between pre-pore state and pore state of tcdA1(<i>2</i>).. 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.]]  
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[[File:ZJU-CHINA_tcdA1_transportation.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.
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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 pH 6. 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).  
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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).  
 
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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).  
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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).  
 
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<h3> RESULTS </h3>
 
<h3> RESULTS </h3>
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<h4> PLASMID CONSTRUCTION </h4>
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<h4> DNA CONSTRUCTION </h4>
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[[File:Digestion_A1%2BpSB1A2.png|200px|thumb|left|Figure 4 digestion confirmation of tcdA1-device in pSB1A2 backbone.]]
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[[File:ZJU-CHINA_CDSA1.png|200px|thumb|left|Figure 4 PCR confirmation results of CDS <i>tcdA1</i>. We choose a 1k fragment in the middle of <i>tcdA1</i> gene as template because the whole gene is much too long.]]
 
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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.
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The template we use is recombinant plasmids.  
 
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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).
 
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It can be clearly seen the two recombinants shown in Figure 4 is two positive cloning.
 
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<h4> PLASMID SEQUNCING </h4>
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<h4> DNA SEQUNCING </h4>
 
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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.
 
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.
 
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<h4> REFERENCE </h4>
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1.  C. Gatsogiannis et al., NATURE 495, 520 (2013-03-20, 2013).
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2.  D. Meusch et al., NATURE 508, 61 (2014).
 
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Latest revision as of 09:39, 18 September 2015

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7035
    Illegal NheI site found at 7368
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 429
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 6469
    Illegal NgoMIV site found at 7129
    Illegal AgeI site found at 2305
  • 1000
    COMPATIBLE WITH RFC[1000]


Characterization

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(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 and confirmed it by digesting and sequencing.


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).. 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 pH 6. 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

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).