Difference between revisions of "Team:Tianjin/Experiment/CFPS"
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<h2><b>Overview</b></h2> | <h2><b>Overview</b></h2> | ||
<p style="font-size:18px">The ability to produce a functional protein in the test tube, rather than in cells, is the essence of cell-free protein synthesis (CFPS)[1].</i> .</p> | <p style="font-size:18px">The ability to produce a functional protein in the test tube, rather than in cells, is the essence of cell-free protein synthesis (CFPS)[1].</i> .</p> | ||
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<p style="font-size:15px"> | <p style="font-size:15px"> | ||
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Fig.1. The basic process of CFPS system | Fig.1. The basic process of CFPS system | ||
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<p style="font-size:18px">The lack of high-throughput approaches for expression and screening of large enzyme libraries remains a | <p style="font-size:18px">The lack of high-throughput approaches for expression and screening of large enzyme libraries remains a | ||
major bottleneck for current enzyme engineering efforts. To address this need, some researchers[2] have developed a high-throughput, fluorescence-based approach for rapid one-pot, microscale expression, and screening of | major bottleneck for current enzyme engineering efforts. To address this need, some researchers[2] have developed a high-throughput, fluorescence-based approach for rapid one-pot, microscale expression, and screening of | ||
different kinds of enzymes. To go further, we try to make our effert to achieve integration of cell-free protein expression with activity screening of enzymes( site-directed mutations of <i>PETase</i>). | different kinds of enzymes. To go further, we try to make our effert to achieve integration of cell-free protein expression with activity screening of enzymes( site-directed mutations of <i>PETase</i>). | ||
</i> .</p> | </i> .</p> | ||
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| + | Fig.2.One-pot approach for integrated expression and activity screening of enzymes | ||
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| + | <h2><b>Background</b></h2> | ||
| + | <p style="font-size:18px">The diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications, such as screeening of enzymes activities. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high-throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology.</p> | ||
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| + | <h2><b>Experiment Design</b></h2> | ||
| + | <p style="font-size:18px">Basically, we utilized the cell-free system to express the enzymes which had been modified in 22 different sites. We conceived that with this method we could acquire the best modifications by screening them in a high-throughput way. Then we used the proteins we got to degrade PET. </p> | ||
| + | <p style="font-size:18px">How to characterize the degradation velocity is the main problem in our scheme. We analyzed the experiment consequences in two ways. For the first one, we rendered the enzymes degrade pNPa, a general substituent for the detection of PET. Then we measured the absorbance of pNP in the optical density of 400 nanometers, which is the degrading product of pNPa. For the second one, we detected the absorbance of MHET in the optical density of 260 nanometers, which is the product in the first step of PET degradation. | ||
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Revision as of 02:12, 5 October 2016
Experiment of CFPS
Overview
The ability to produce a functional protein in the test tube, rather than in cells, is the essence of cell-free protein synthesis (CFPS)[1]. .
Fig.1. The basic process of CFPS system
The lack of high-throughput approaches for expression and screening of large enzyme libraries remains a major bottleneck for current enzyme engineering efforts. To address this need, some researchers[2] have developed a high-throughput, fluorescence-based approach for rapid one-pot, microscale expression, and screening of different kinds of enzymes. To go further, we try to make our effert to achieve integration of cell-free protein expression with activity screening of enzymes( site-directed mutations of PETase). .
Fig.2.One-pot approach for integrated expression and activity screening of enzymes
Background
The diversity of the cell-free systems allows in vitro synthesis of a wide range of proteins for a variety of downstream applications, such as screeening of enzymes activities. In the post-genomic era, cell-free protein synthesis has rapidly become the preferred approach for high-throughput functional and structural studies of proteins and a versatile tool for in vitro protein evolution and synthetic biology.
Experiment Design
Basically, we utilized the cell-free system to express the enzymes which had been modified in 22 different sites. We conceived that with this method we could acquire the best modifications by screening them in a high-throughput way. Then we used the proteins we got to degrade PET.
How to characterize the degradation velocity is the main problem in our scheme. We analyzed the experiment consequences in two ways. For the first one, we rendered the enzymes degrade pNPa, a general substituent for the detection of PET. Then we measured the absorbance of pNP in the optical density of 400 nanometers, which is the degrading product of pNPa. For the second one, we detected the absorbance of MHET in the optical density of 260 nanometers, which is the product in the first step of PET degradation.
