Team:Tec-Monterrey/projectoverview
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Inverted sugar is conventionaly synthetizing by the acid hydrolysis of sucrose. However, such reaction has a low conversion efficiency and high-energy consumption (¿.....?). As an alternative method to the traditional chemical process to produce inverted sugar, cell surface display was suggested. The cell surface display is a technique to display proteins on the surface of bacteria, fungi, or mammalian cells by fusing them to surface anchoring motifs. This technique has a wide range of biotechnological and industrial applications, including development of vaccines, peptide and antibody libraries, bioremediation, whole-cell-biosensors, and whole-cell-biocatalysis. When proteins are expressed in the outer membrane of <i>Escherichia coli</i> the cell envelope acts as their matrix. This display is achievable thanks to several displaying systems as outer membrane porins, lipoproteins, GPI-anchored-proteins, fimbriae, and autotransporters. (Jana S & Deb JK, 2005; Lee SH <i>et al</i>., 2004) Displaying proteins on the cell surface also makes preparing or purifying the protein unnecessary in many instances. Whole cells displaying the molecule of interest can be used in industrial process reactions or analytical assays and then can be simply recovered by centrifugation. (Joachim J & Meyer TF, 2007) | Inverted sugar is conventionaly synthetizing by the acid hydrolysis of sucrose. However, such reaction has a low conversion efficiency and high-energy consumption (¿.....?). As an alternative method to the traditional chemical process to produce inverted sugar, cell surface display was suggested. The cell surface display is a technique to display proteins on the surface of bacteria, fungi, or mammalian cells by fusing them to surface anchoring motifs. This technique has a wide range of biotechnological and industrial applications, including development of vaccines, peptide and antibody libraries, bioremediation, whole-cell-biosensors, and whole-cell-biocatalysis. When proteins are expressed in the outer membrane of <i>Escherichia coli</i> the cell envelope acts as their matrix. This display is achievable thanks to several displaying systems as outer membrane porins, lipoproteins, GPI-anchored-proteins, fimbriae, and autotransporters. (Jana S & Deb JK, 2005; Lee SH <i>et al</i>., 2004) Displaying proteins on the cell surface also makes preparing or purifying the protein unnecessary in many instances. Whole cells displaying the molecule of interest can be used in industrial process reactions or analytical assays and then can be simply recovered by centrifugation. (Joachim J & Meyer TF, 2007) | ||
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- | The Tat (twin-arginine translocation) system functions to translocate folded proteins across the membrane while the Sec secretory pathway translocates unfolded substrates. The Sec translocase is comprised of the SecYEG translocation channel and the accessory components SecA, SecDFYajC, and YidC. | + | The Tat (twin-arginine translocation) system functions to translocate folded proteins across the membrane while the Sec secretory pathway translocates unfolded substrates. The Sec translocase is comprised of the SecYEG translocation channel and the accessory components SecA, SecDFYajC, and YidC. (Yuan J <i>et al</i>., 2010 & Yuan J <i>et al </i>., 2010) Both the type II secretory system and the type V autotransporter system are natural translocation system to import/export substrates through the periplasm and membrane. The type II secretory system can take both Tat and Sec system while the type V autotransporters use the Sec system because they are composed of a N-terminal Sec-dependent signal peptide, a passenger domain, and a translocator domain that is predicted to form a β-barrel. (Rutherford <i>et al</i>., 2006) Both lpp and phoA signal peptides are natural Sec-dependent <i>E. coli</i> signal peptides which permits the translocation of the outer membrane proteins by the type II and the type V system, respectively. |
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- | <p class="textojustif"> In this project, the natural passenger domain of the autotransporter estA from <i>Pseudomonas sp </i> was replaced by a cellulase (CelD) and an invertase (SacC) as means to display them at the bacterial surface by the translocator domain of the estA protein. | + | <p class="textojustif"> In this project, the natural passenger domain of the autotransporter estA from <i>Pseudomonas sp </i> was replaced by a cellulase (CelD) and an invertase (SacC) as means to display them at the bacterial surface by the translocator domain of the estA protein. Also, these enzymes were fused with an integral outer membrane fragment of ompA protein (BBa_K103006) to display them at the outer membrane of <i>E. coli</i>. Using the signal peptide of a protein which is naturally transported to the cytoplasma (signal peptide of phoA and lpp), we intend to export CelD and SacC to the external surface of <i>E. coli</i>. |
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- | Using the signal peptide of a protein which is naturally transported to the cytoplasma (signal peptide of | + | |
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The sequence of CelD used to construct genetic frame was modified according to Chauvaux <i>et al</i>., substituing Asp523 by Ala, since that mutation increases specific activity of CelD to 224% . (Chauvaux S et al., 1992) | The sequence of CelD used to construct genetic frame was modified according to Chauvaux <i>et al</i>., substituing Asp523 by Ala, since that mutation increases specific activity of CelD to 224% . (Chauvaux S et al., 1992) | ||
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- | Our new genetic construct will be able to immobilize invertase (sacC) on the outer membrane of <i>E. coli</i>, fusing the enzyme with fragments of ompA and estA. This chassis will be capable of transforming sucrose into fructose without further primary recovery, purification, | + | Our new genetic construct will be able to immobilize an invertase (sacC) and on the outer membrane of <i>E. coli</i>, fusing the enzyme with fragments of ompA and estA. This chassis will be capable of transforming sucrose into fructose without further primary recovery, purification, or immobilization steps to obtain enzymes; thus reducing the amount of unit operations and cutting production costs. At the same time, we will immobilize cellulase (celD) with the same strategy to take advantage of cellulose residues from the sugarcane process, making the process more sustainable. |
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