Team:Michigan/Design/

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<h1><font size="10" <em class = "colored">Design: Our Expression Vector</em></font></h1></p><font size="3"><font color="#191970"> 2014 Michigan Synthetic Biology Team </font></font color></p>
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<h1><font size="10"> Project Design</font></h1></p><font size="3"><font color="#191970"> University of Michigan iGEM Team </font></font color></p>
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<p style="position:absolute;height:200px;left:0px;top:200px">Our designed construct consists of a secretion tag, OsmY, at the N-terminus followed by a His-Tag, TEV cleavage site and the desired protein at the C-terminus. 
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<p style="position:absolute;height:200px;left:0px;top:248px">As the construct gets translated, the signal sequence of OsmY will signal the cell to carry the ribosome at the inner membrane (Qian et al. 2008). The remaining of the construct will be translated into the periplasm, allowing the protein of interest to fold in an oxidative environment and form disulfide bonds (Qian et al. 2008). Further on, the OsmY-tag will carry the construct out of the periplasm into the media through an unknown mechanism (Qian et al. 2008).
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<p style="position:absolute;height:200px;left:0px;top:730px">Once outside the cell, the protein can be harvested simply by centrifuging the growth media to separate the cells from the extracellular proteins. The supernatant is then run through a nickel column, to which the construct will bind due to the His-Tag, therefore separating the desired protein from extracellular contaminants. After eluting the construct from the column, TEV-His protease (a TEV protease linked to a poly-histidine tag) is added to the solution in order to cleave off the desired protein. After cleavage, the solution is run through a nickel column once more; the TEV-His and the construct fragment containing the tags will bind to the column while our desired protein run directly through the column without binding.</p>
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<img src="https://static.igem.org/mediawiki/2014/0/04/OsmY_purification_system_with_bubbles.png" style="position:absolute;top:856px;left:200px;width:500px">
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<p style="position:absolute;height:200px;left:0px;top:1350px">In this project, we’ve used an antibody scFv as our protein of interest. To create a single chain scFv we’ve inserted the sequence of a heavy variable chain upstream of the sequence of a complementary light variable chain and linked them with a DNA fragment encoding a Myc tag (Thie et al. 2008). The Myc tag not only allowed spacing between the 2 antibody fragment but also enabled detection of the scFv (Thie et al. 2008)</p>
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<p style="position:absolute;height:200px;left:0px;top:1550px">[1]Qian, Z.-G. G., Xia, X., Choi, J. H. & my Lee, S. Y. Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli. Biotechnology and bioengineering 101, 587–601 (2008).
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</br>
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[2]Thie, H., Schirrmann, T., Paschke, M., Dübel, S. & Hust, M. SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli. New biotechnology 25, 49–54 (2008).
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</br>
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[3]Röthlisberger, D., Pos, K. M. & Plückthun, A. An antibody library for stabilizing and crystallizing membrane proteins - selecting binders to the citrate carrier CitS. FEBS Letters 564, 340–8 (2004).
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</br>
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[4]Cheng, C.-M. et al. Functional Production of a Soluble and Secreted Single-Chain Antibody by a Bacterial Secretion System. PLoS ONE 9, e97367 (2014).
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</br>
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[5]Meyer, T. et al. Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display. BMC Biotechnology 12, 29 (2012).
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</br>
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[6]Bokinsky, G. et al. Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 108, 19949–54 (2011).
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Latest revision as of 03:31, 18 October 2014

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Our designed construct consists of a secretion tag, OsmY, at the N-terminus followed by a His-Tag, TEV cleavage site and the desired protein at the C-terminus.

As the construct gets translated, the signal sequence of OsmY will signal the cell to carry the ribosome at the inner membrane (Qian et al. 2008). The remaining of the construct will be translated into the periplasm, allowing the protein of interest to fold in an oxidative environment and form disulfide bonds (Qian et al. 2008). Further on, the OsmY-tag will carry the construct out of the periplasm into the media through an unknown mechanism (Qian et al. 2008).

Once outside the cell, the protein can be harvested simply by centrifuging the growth media to separate the cells from the extracellular proteins. The supernatant is then run through a nickel column, to which the construct will bind due to the His-Tag, therefore separating the desired protein from extracellular contaminants. After eluting the construct from the column, TEV-His protease (a TEV protease linked to a poly-histidine tag) is added to the solution in order to cleave off the desired protein. After cleavage, the solution is run through a nickel column once more; the TEV-His and the construct fragment containing the tags will bind to the column while our desired protein run directly through the column without binding.

In this project, we’ve used an antibody scFv as our protein of interest. To create a single chain scFv we’ve inserted the sequence of a heavy variable chain upstream of the sequence of a complementary light variable chain and linked them with a DNA fragment encoding a Myc tag (Thie et al. 2008). The Myc tag not only allowed spacing between the 2 antibody fragment but also enabled detection of the scFv (Thie et al. 2008)

[1]Qian, Z.-G. G., Xia, X., Choi, J. H. & my Lee, S. Y. Proteome-based identification of fusion partner for high-level extracellular production of recombinant proteins in Escherichia coli. Biotechnology and bioengineering 101, 587–601 (2008).
[2]Thie, H., Schirrmann, T., Paschke, M., Dübel, S. & Hust, M. SRP and Sec pathway leader peptides for antibody phage display and antibody fragment production in E. coli. New biotechnology 25, 49–54 (2008).
[3]Röthlisberger, D., Pos, K. M. & Plückthun, A. An antibody library for stabilizing and crystallizing membrane proteins - selecting binders to the citrate carrier CitS. FEBS Letters 564, 340–8 (2004).
[4]Cheng, C.-M. et al. Functional Production of a Soluble and Secreted Single-Chain Antibody by a Bacterial Secretion System. PLoS ONE 9, e97367 (2014).
[5]Meyer, T. et al. Identification of immunogenic proteins and generation of antibodies against Salmonella Typhimurium using phage display. BMC Biotechnology 12, 29 (2012).
[6]Bokinsky, G. et al. Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 108, 19949–54 (2011).

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