Team:NEAU-Harbin/link1.html

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<h3 style="padding-left:20px">1.Fusion protein and linkers </h3>
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<p style="padding-left:20px">Agrobacterium-mediated transformation method</p>
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<p style="padding-left:20px">Fusion protein was a genetic expression product, which was composed by two or more head-to-tail ligation coding region that were controlled by the same regulatory sequence. The design of linker played an important part in the fusion protein with connecting peptides.<p>
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<p style="padding-left:20px">Agrobacterium-mediated fungal transformation (AMT) is a potential tool for performing targeted and random mutagenesis .This method is commonly used for plant-cell transformation and is recently widely applied to various fungi. Through AMT system, T-DNA can be transferred to a wide variety of fungi with high efficiency. </p>
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<p style="padding-left:20px">The advantages of fusion enzymes lie in that it is able to integrate a variety of active catalytic enzyme in a protein. In the process of a large number of sequential enzyme catalysis, the product of an enzyme (intermediate) is the substrate of next step of enzyme catalytic reaction, and these enzymes are often in a free state especially in plant cells, so the distance between the enzyme molecule is difficult to control, which restricts its catalytic efficiency. Our solution is to connect multiple lysosomal enzymes through the fusion protein technology and put them in the sequence of the catalytic reaction to adjust the space structure to the effect, thus we can control of reaction efficiency.</p>
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<p style="text-align:center"><img src="http://nd.cn.usa.wakelion.net/project/link1.jpg"></p>
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<p style="padding-left:20px">Peptide linkers are used to connect protein domains. For this connection it is important that the linker itself has no influence on the connected parts. Therefore the sequence of the linker is designed for amino acids which do not interact with other amino acid residues. The amino acids glycine and serine are zwitterionic and hydrophile, these properties make them a good choice for the repetitive sequence of the linker.</p>
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<p style="padding-left:20px">The length of the linker is important to guarantee the independent function of two connected parts. When the linker is too short there might be a sterical interference between the parts and if it is too long, the construct can be instable. Also, it is important that the linker has a certain flexibility.</p>  
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<p style="padding-left:20px">（more）: </p>
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<p style="padding-left:20px">Structure of <i>A. tumefaciens</i>' plasmid </p>
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<p style="padding-left:20px">• The gram-negative bacterium <i>A. tumefaciens</i> is a plant pathogen, which causes crown gall tumors.<i>A. tumefaciens</i> induces this tumorous growth by transferring a part of its DNA (T-DNA) which is located on its 200-kbp tumor-inducing (Ti) plasmid to the host. After integration into the host genome, genes that are naturally located on this T-DNA and encode enzymes for the production of plant growth regulators are expressed.</p>
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<p style="padding-left:20px">• And another segment, the virulence region, which is composed of a large number of vir genes, is necessary for the tumorigenicity. Proteins encoded by the virulence region are involved in the formation, transport and possibly also integration of the T-DNA. And the T-region of the Ti plasmid is surrounded by a 24-bp border repeat, which is the cis-acting signal for the DNA delivery system to plant cells. Otherwise, all the sequences of the natural T-DNA can be deleted and replaced by other DNA sequences without a negative effect. So the binary vector system is used, in which the T-DNA and the virulence region are placed on two separate plasmids.</p>
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<p style="padding-left:20px">• mechanism</p>
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<p style="padding-left:20px">• Phenolic compounds such as acetosyringone are used to induce the vir genes that encode the T-DNA transfer machinery of <i>A. Tumefaciens</i>. VirA, an inner membrane protein, senses acetosyringone and responds by autophosphorylation.</p>
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<p style="padding-left:20px">• The chromosomally encoded protein, ChvE,interacts with the VirA protein to further enhance levels of vir induction in the presence of specific monosaccharides.</p>
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<p style="padding-left:20px">• The activated VirG, which has DNA-binding properties, then acts as a transcriptional activator of itself and other virulence genes after VirA transfers phosphoryl group to it.</p>
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<p style="padding-left:20px">• For the generation of a single-stranded DNA copy of the T-DNA,the virC and virD operons are needed.VirC1 can bind the25-bp “overdrive” sequence and thereby</p>
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<p style="padding-left:20px">• Stimulates T-strand production. The VirD2 protein, assisted by VirD1, stays covalently attached to the 5’ end of the T-strand.</p>
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<p style="padding-left:20px">• The next step in T-DNA transfer is piloting the T-strand through the bacterial membrane and cell wall. The VirB proteins form a transport pore and a structure on the surface, and the virulence proteins VirE2, VirE3, and VirF are also exported .VirE2 is a single-stranded DNA-binding protein and is thought to coat the T-strand in the host to protect it against nucleases and to keep the T-strand in an unfolded state to facilitate transport. Once inside the nucleus, the T-DNA stably integrates into the genome.</p>
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<p style="padding-left:20px">• advantages</p>
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<p style="padding-left:20px">• It has been shown to have several advantages over conventional transformation methods. AMT generates a high percentage of transformants with a single-copy integrated DNA, which facilitates the isolation of tagged genes, and the T-DNA is an efficient substrate for homologous recombination. Above all, AMT is well suited to perform insertional mutagenesis in fungi.</p>
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<p style="padding-left:20px">Based on these rules we designed our linkers to make the fusion gene crtEBI and crtYZW. The sequence of the linker is: GGTAGCGGCTCCGGAAGCGGTTCCGGCAGC.</p>
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Latest revision as of 12:19, 15 October 2014

Sponsor

Agrobacterium-mediated transformation method

Agrobacterium-mediated fungal transformation (AMT) is a potential tool for performing targeted and random mutagenesis .This method is commonly used for plant-cell transformation and is recently widely applied to various fungi. Through AMT system, T-DNA can be transferred to a wide variety of fungi with high efficiency.

（more）:

Structure of A. tumefaciens' plasmid

• The gram-negative bacterium A. tumefaciens is a plant pathogen, which causes crown gall tumors.A. tumefaciens induces this tumorous growth by transferring a part of its DNA (T-DNA) which is located on its 200-kbp tumor-inducing (Ti) plasmid to the host. After integration into the host genome, genes that are naturally located on this T-DNA and encode enzymes for the production of plant growth regulators are expressed.

• And another segment, the virulence region, which is composed of a large number of vir genes, is necessary for the tumorigenicity. Proteins encoded by the virulence region are involved in the formation, transport and possibly also integration of the T-DNA. And the T-region of the Ti plasmid is surrounded by a 24-bp border repeat, which is the cis-acting signal for the DNA delivery system to plant cells. Otherwise, all the sequences of the natural T-DNA can be deleted and replaced by other DNA sequences without a negative effect. So the binary vector system is used, in which the T-DNA and the virulence region are placed on two separate plasmids.

• mechanism

• Phenolic compounds such as acetosyringone are used to induce the vir genes that encode the T-DNA transfer machinery of A. Tumefaciens. VirA, an inner membrane protein, senses acetosyringone and responds by autophosphorylation.

• The chromosomally encoded protein, ChvE,interacts with the VirA protein to further enhance levels of vir induction in the presence of specific monosaccharides.

• The activated VirG, which has DNA-binding properties, then acts as a transcriptional activator of itself and other virulence genes after VirA transfers phosphoryl group to it.

• For the generation of a single-stranded DNA copy of the T-DNA,the virC and virD operons are needed.VirC1 can bind the25-bp “overdrive” sequence and thereby

• Stimulates T-strand production. The VirD2 protein, assisted by VirD1, stays covalently attached to the 5’ end of the T-strand.

• The next step in T-DNA transfer is piloting the T-strand through the bacterial membrane and cell wall. The VirB proteins form a transport pore and a structure on the surface, and the virulence proteins VirE2, VirE3, and VirF are also exported .VirE2 is a single-stranded DNA-binding protein and is thought to coat the T-strand in the host to protect it against nucleases and to keep the T-strand in an unfolded state to facilitate transport. Once inside the nucleus, the T-DNA stably integrates into the genome.

• advantages

• It has been shown to have several advantages over conventional transformation methods. AMT generates a high percentage of transformants with a single-copy integrated DNA, which facilitates the isolation of tagged genes, and the T-DNA is an efficient substrate for homologous recombination. Above all, AMT is well suited to perform insertional mutagenesis in fungi.

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