Difference between revisions of "Team:TU Darmstadt/Project/Bio/InVitroDegradation/sec2"
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− | <p>1) To check the binding to a silica-surface and stability of boundary we fused the Si4-tag by iGEM Leeds 2013 (2) with GFP (<a href=" http://parts.igem.org/Part:BBa_E0040" title="Opens internal link in current window" class="internal link">BBa_E0040</a>), YFP (BBa_E0030) and CFP (BBa_E0020) using Overlap PCR / Biobrick Assembly. After Expression and Purification we want to visualize the boundary to a silica-coated surface in different buffer conditions and time dependency off the boundary.</p> | + | <p>1) To check the binding to a silica-surface and stability of boundary we fused the Si4-tag by iGEM Leeds 2013 (2) with GFP (<a href=" http://parts.igem.org/Part:BBa_E0040" title="Opens internal link in current window" class="internal link">BBa_E0040</a>), YFP (<a href=" http://parts.igem.org/Part:BBa_E0030" title="Opens internal link in current window" class="internal link">BBa_E0030</a>) and CFP (<a href=" http://parts.igem.org/Part:BBa_E0020" title="Opens internal link in current window" class="internal link">BBa_E0020</a>) using Overlap PCR / Biobrick Assembly. After Expression and Purification we want to visualize the boundary to a silica-coated surface in different buffer conditions and time dependency off the boundary.</p> |
<p>2) By linking the zinc finger ligand sequence to GFP (BBa_E0040), YFP (BBa_E0030) and CFP (BBa_E0020) and combining purified protein scaffold domains with the fluorescent proteins we want to control the boundary of ligand scaffold domain in different buffer conditions.</p> | <p>2) By linking the zinc finger ligand sequence to GFP (BBa_E0040), YFP (BBa_E0030) and CFP (BBa_E0020) and combining purified protein scaffold domains with the fluorescent proteins we want to control the boundary of ligand scaffold domain in different buffer conditions.</p> | ||
<p>3) To verify the advantage of our protein scaffolding we want to compare the yield of degrading xylan only based on the three degradation enzymes in one composite to the combination of the composite and the protein-scaffold.</p> | <p>3) To verify the advantage of our protein scaffolding we want to compare the yield of degrading xylan only based on the three degradation enzymes in one composite to the combination of the composite and the protein-scaffold.</p> |
Revision as of 18:10, 17 September 2015
Contents
Immobilized protein scaffold
Abstract
Introduction
Experimental setup
1) binding efficiency of the silica tag to the silica surface and stability of boundary
2) binding efficiency of the enzyme-scaffold interaction sequence
3) Xylan degradation yield enrichment using the protein scaffold
1) To check the binding to a silica-surface and stability of boundary we fused the Si4-tag by iGEM Leeds 2013 (2) with GFP (BBa_E0040), YFP (BBa_E0030) and CFP (BBa_E0020) using Overlap PCR / Biobrick Assembly. After Expression and Purification we want to visualize the boundary to a silica-coated surface in different buffer conditions and time dependency off the boundary.
2) By linking the zinc finger ligand sequence to GFP (BBa_E0040), YFP (BBa_E0030) and CFP (BBa_E0020) and combining purified protein scaffold domains with the fluorescent proteins we want to control the boundary of ligand scaffold domain in different buffer conditions.
3) To verify the advantage of our protein scaffolding we want to compare the yield of degrading xylan only based on the three degradation enzymes in one composite to the combination of the composite and the protein-scaffold.
Results
In vitro bioreactor
- J. E. Dueber et al., Synthetic protein scaffolds provide modular control over metabolic flux. Nat Biotechnol 27, 753-759 (2009).
- R. R. Naik, L. L. Brott, S. J. Clarson, M. O. Stone, Silica-precipitating peptides isolated from a combinatorial phage display peptide library. J Nanosci Nanotechnol 2, 95-100 (2002).
- R. J. Conrado et al., DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic Acids Res 40, 1879-1889 (2012).