Team:Korea U Seoul/Project/sub desc

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                   Background(OVERVIEW)
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    Enzyme purification is costly, and purified enzymes cannot be used multiple times due to decrease in activity after few reactions. To overcome this problem, ‘whole-cell biocatalysis’, which uses the microorganism itself as a catalyst, was introduced. Related to this, ‘Cell surface display’ can provide higher rate of access to the substrate, but due to the possibility of toxicity, its result has a low rate of actual expression rate. To make more efficient and stable surface display system,“sortase-based surface display at gram-positive bacteria” has been researched in many fields lately. Sortase functions as ‘protein ligase’ that recognizes specific amino acids sequence(LPXT) of each peptides and links them together. It is also associated with cell wall-bound protein and pili synthesis. ‘Sortase-based surface display’ can induce stable enzyme surface display by reducing cell toxicity and holding enzymes tightly to the cell wall with covalent bond.<br />
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    Sortases, enzymes that recognize and cleave the specific sorting signal of secreted proteins to form isopeptide bonds between the secreted proteins and polypeptides, function as protein ligase to form the cell-wall surface of gram-positive bacteria. In case of <i>C.diphtheriae</i>, which belongs to the same genus of our experimental bacteria <i>Corynebacterium glutamicum</i>, has total of 6 sortases, 5 pilus specific sortases (Srt A,B,C,D,E) and 1 housekeeping sortase (Srt F) involved in the formation of all types of pili. <br />
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However, it cannot overcome low display yield and it should include an extra step of treating sortase in vitro. Therefore, referring to sortase-based study, we are going to make enzyme polymer (enzyme pili) on cell wall by modifying pili units used in pili synthesis in other species in Corenebacterium genus. This study provides more efficient way of enzymatic process and application of pili biosynthesis
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There are 9 types of pillins that comprise pilus; Spa A, B, C, D, E, F, G, H, I, J. SrtA build up SpaA-type pili, which is composed of Spa A, B and C. Srt B/C and Srt d/E each forms SpaD-type pili and SpaH-type pili, which is made up of Spa D, E, F and Spa H, I, J respectively. Like this, the names of each pili types are generally from the names of many Spa proteins. <br />
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The picture below shows the process of SpaA type pili formation, using SrtA. Sortase class A enzymes recognize the sequence LPXTG at the carboxyl terminus of surface protein precursors. Cystein of SrtA recognizes LPXTG motif of SpaC, cleaves between T and G, forming SpaC-SrtA intermediate via nucleophilic attack. This intermediate is again attacked by lysine of SrtA bounded SpaA, and the process is continued to form SpaC–SpaAn–SrtA intermediates.<br />
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Nucleophilic attack by Lysine of SrtF bounded SpaB form SpaC-SpaA(n)-SpaB-SrtF intermediate. The product of this SrtA reaction is covalently linked to lipidⅡ and is then incorporated into the cell wall envelope, terminating the formation of SpaA-type pilus.
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(Architects at the bacterial surface — sortases and the assembly of pili with isopeptide bonds <i>Antoni P. A. Hendrickx, Jonathan M. Budzik, So-Young Oh and Olaf Schneewind</i>)
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                Our Team project is making pearl by E.coli induced Nacrein gene. At first, amplify kinds of nacrein genes by DH5a. Next, transformation of these genes to BW25113, BL21(DE3). Then nacrein genes make nacrein proteins. Finally they make nacrein aragonite structures itself and they forms the pearl.<br /><br />
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The main objective of our project is to construct a novel “protein whip” platform, with which we can make <i>Corynebacterium glutamicum</i> to express other corynebacterium’s pili structure comprised of chains of a protein of our choice. As our first try, we decided to make pili made out of green fluorescence proteins (GFP); in order to do so, we substituted SpaA protein, one of the surface proteins in the Pilin A gene cluster, into green fluorescence protein, and transformed a vector containing the modified Pilin A gene cluster into a <i>C. glutamicum</i> strain. <br />Our “protein whip” platform is expected to have many practical applications. For example, pili made out of an enzyme, enzyme whip will enable the reaction to take place with high efficiency, for a great number of the enzyme included in the pili will be able to “attack” the reactants simultaneously. Biofilms made of strains of bacteria that express pili comprised of chains of specific amino acids such as histidine or cysteine that readily bind to heavy metals may be utilized to purify water contaminated with heavy metals. <br />
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                Nacrein has carbonic anhydrase domain that can make CO2 to carbonic acid, and it changes to bicarbonate ion(HCO3-) and H+. And We use Nacrein, Nacrein CA, Nacrein DR, Nacrein R gene to check what gene can make lots of pearl. (CA, DR and R indicate first carbonic anhydrase domain clone, repeat deleted-clone and only repeat clone, respectively.) We can make pearl with reducing CO2.<br /><br />
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Having a number of potential applications is not the sole merit of our project; by using <i>C. glutamicum</i> instead of widely exploited <i>Escherichia coli</i>, our project also contributes to expanding model organisms used in synthetic biology beyond <i>E. coli</i>.
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                The Korea_U_Seoul team aims to design Pearl-coli that is <i>E.coli</i> able to convert atmospheric CO2 into a pearl powder material. The design is based on cell surface display of nacrein in <i>E.coli</i>. Nacrein is a major protein component in nacre (an organic-inorganic composite layer found in outer coating of pearls). We divided nacrein into several functional regions - carbonic anhydrase (CA), calcium binding and scaffold repeats. Carbonic anhydrase domain fixes CO2 into carbonic acid changing to bicarbonate ion in aqueous solution. We assume that the other regions except CA is related to formation of nacre layer in a pearl. The whole nacrein and those functional regions (CA, NA, R) are displayed in cell surface of <i>E.coli</i>. We will examine if displayed nacrein in <i>E.coli</i> can make a pearl powder in a solution or fabricate a nacre-like structure while atmospheric CO2 is fixed into bicarbonate. Once a nacre material can be prepared from Pearl-coli, we will grow <i>E.coli</i> in a confined container to make synthetic pearl. The Pearl-coli has dual-function such as (1) mitigate the global warming by CO2 reduction, (2) prepare valuable pearl-like raw materials.  
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Latest revision as of 00:48, 18 October 2014

Background

Sortases, enzymes that recognize and cleave the specific sorting signal of secreted proteins to form isopeptide bonds between the secreted proteins and polypeptides, function as protein ligase to form the cell-wall surface of gram-positive bacteria. In case of C.diphtheriae, which belongs to the same genus of our experimental bacteria Corynebacterium glutamicum, has total of 6 sortases, 5 pilus specific sortases (Srt A,B,C,D,E) and 1 housekeeping sortase (Srt F) involved in the formation of all types of pili.
There are 9 types of pillins that comprise pilus; Spa A, B, C, D, E, F, G, H, I, J. SrtA build up SpaA-type pili, which is composed of Spa A, B and C. Srt B/C and Srt d/E each forms SpaD-type pili and SpaH-type pili, which is made up of Spa D, E, F and Spa H, I, J respectively. Like this, the names of each pili types are generally from the names of many Spa proteins.
The picture below shows the process of SpaA type pili formation, using SrtA. Sortase class A enzymes recognize the sequence LPXTG at the carboxyl terminus of surface protein precursors. Cystein of SrtA recognizes LPXTG motif of SpaC, cleaves between T and G, forming SpaC-SrtA intermediate via nucleophilic attack. This intermediate is again attacked by lysine of SrtA bounded SpaA, and the process is continued to form SpaC–SpaAn–SrtA intermediates.
Nucleophilic attack by Lysine of SrtF bounded SpaB form SpaC-SpaA(n)-SpaB-SrtF intermediate. The product of this SrtA reaction is covalently linked to lipidⅡ and is then incorporated into the cell wall envelope, terminating the formation of SpaA-type pilus.

(Architects at the bacterial surface — sortases and the assembly of pili with isopeptide bonds Antoni P. A. Hendrickx, Jonathan M. Budzik, So-Young Oh and Olaf Schneewind)

Description

The main objective of our project is to construct a novel “protein whip” platform, with which we can make Corynebacterium glutamicum to express other corynebacterium’s pili structure comprised of chains of a protein of our choice. As our first try, we decided to make pili made out of green fluorescence proteins (GFP); in order to do so, we substituted SpaA protein, one of the surface proteins in the Pilin A gene cluster, into green fluorescence protein, and transformed a vector containing the modified Pilin A gene cluster into a C. glutamicum strain.
Our “protein whip” platform is expected to have many practical applications. For example, pili made out of an enzyme, enzyme whip will enable the reaction to take place with high efficiency, for a great number of the enzyme included in the pili will be able to “attack” the reactants simultaneously. Biofilms made of strains of bacteria that express pili comprised of chains of specific amino acids such as histidine or cysteine that readily bind to heavy metals may be utilized to purify water contaminated with heavy metals.
Having a number of potential applications is not the sole merit of our project; by using C. glutamicum instead of widely exploited Escherichia coli, our project also contributes to expanding model organisms used in synthetic biology beyond E. coli.

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