Difference between revisions of "Part:BBa K1668005"

 
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<partinfo>BBa_K1668005 short</partinfo>
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<partinfo>BBa_K166805 short</partinfo>
  
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metK is the S-adenosylmethionine synthetase gene from Streptomyces avermitilis, which was found to stimulate the production of avermectins. When wild-type strain ATCC31267 was transformed with pYJ02 and pYJ03, two metK expression plasmids, avermectin production was increased about 2.0-fold and 5.5-fold compared with that in the control strains, respectively. The avermectin productivity of each culture was quantitatively measured by HPLC analysis.  
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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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As the kinetic study revealed, avermectin overproduction in recombinant strain was not caused by the change of cell growth rate or copy number effect. Instead, metK stimulates the avermectin production by increasing the intracellular concentration of S-adenosylmethionine (SAM), an important intermediate product in avermectin production. However, there may be a maximum concentration of SAM for the production of avermectin in S. avermitilis, over which it has no any effect on the antibiotic production.
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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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The results of experiments showed that there were different effects of metK expression levels on avermectin production in various S. avermitilis strains. The gene expression levels of metK in two engineered strain, GB-165 and 76-05, were much higher then those in wild-type strain, whereas the avermectin productivity in these two strains were not significantly improved. It probably because the high expression level of metK limited the increasement of avermectin by overexpression of metK.
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===Usage and Biology===
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K1668005 parameters</partinfo>
 
<partinfo>BBa_K1668005 parameters</partinfo>
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<h2>'''Characterization'''</h2>
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<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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We clone and standardize the gene into standard plasmid pSB1C3 and confirmed it by digesting and sequencing.
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<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: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.]]
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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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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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<h3> RESULTS </h3>
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<h4> PLASMID 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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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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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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<h4> PLASMID 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.
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Revision as of 12:02, 16 September 2015

No part name specified with partinfo tag.

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, 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

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 7.5k part shows 100% agreement with the desired sequence.