Engineering bioorthogonal reactions on proteins in live cells Public Deposited

http://ir.library.oregonstate.edu/concern/undergraduate_thesis_or_projects/8p58pf593

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  • This project deals with a method to optimize in vivo labeling using small fluorescent molecules via bioorthogonal reactions. The reaction used involves our unnatural amino acid 4-(6-methyl-s-tetrazin-3-yl)aminophenylalanine (AMT-Phe). The amino acid is site-specifically incorporated into GFP and then reacted with a labeled, strained trans-cyclooctene, resulting in a labeled protein. However we have noticed that the label becomes detached from the protein. The conditions and influential factors that cause this are unknown, so tests to determine the stability of AMT-Phe were run using mass spectrometry. Results have shown that AMT-Phe might be decomposing once incorporated in the protein over time, suggesting that it’s not very stable. AMT-Phe contains tetrazine functionality, with a methyl group attached to the tetrazine. A potentially more stable version of AMT-Phe is to replace the methyl group with a phenyl group. This potentially more stable and reactive amino acid, 4-(6-methyl-s-tetrazin-3-yl)aminophenyl- alanine (APT-Phe), was synthesized, and a series of tests on incorporation and reactive rates were run to compare with AMT-Phe. Although APT-Phe didn’t incorporate into GFP very well, preliminary data shows that it has a 6-fold faster reaction rate than AMT-Phe.
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  • Original submission was .pptx created with Microsoft PowerPoint for PC 2010, version 14 and corrected with Microsoft PowerPoint for Mac 2011, version 14.3.1. File was converted to .pdf using Microsoft PowerPoint for Mac 2011, version 14.3.1.
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  • description.provenance : Approved for entry into archive by Deanne Bruner(deanne.bruner@oregonstate.edu) on 2013-05-29T18:08:25Z (GMT) No. of bitstreams: 3 license_rdf: 1089 bytes, checksum: 0a703d871bf062c5fdc7850b1496693b (MD5) Machovina CUE poster.pdf: 1583296 bytes, checksum: e87b076ee2a28193a1feeee9f384ae9b (MD5) Machovina CUE poster.pptx: 870224 bytes, checksum: f64beaad037620072c4268ceb8f07996 (MD5)
  • description.provenance : Made available in DSpace on 2013-05-29T18:08:25Z (GMT). No. of bitstreams: 3 license_rdf: 1089 bytes, checksum: 0a703d871bf062c5fdc7850b1496693b (MD5) Machovina CUE poster.pdf: 1583296 bytes, checksum: e87b076ee2a28193a1feeee9f384ae9b (MD5) Machovina CUE poster.pptx: 870224 bytes, checksum: f64beaad037620072c4268ceb8f07996 (MD5)
  • description.provenance : Submitted by Melodie Machovina (machovim@onid.orst.edu) on 2013-05-29T01:05:09Z No. of bitstreams: 3 license_rdf: 1089 bytes, checksum: 0a703d871bf062c5fdc7850b1496693b (MD5) Machovina CUE poster.pdf: 1583296 bytes, checksum: e87b076ee2a28193a1feeee9f384ae9b (MD5) Machovina CUE poster.pptx: 870224 bytes, checksum: f64beaad037620072c4268ceb8f07996 (MD5)

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