Change search
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Improving unnatural amino acid mutagensis efficiency and selectivity in mammalian cell
KTH, School of Biotechnology (BIO).
2016 (English)Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE creditsStudent thesis
Abstract [en]

Genetically encoded, site-specific incorporation of unnatural amino acids (UAA)into proteins through selective recoding of an amber stop codon provides apowerful route for expressing synthetic proteins in living cells. Recoding of theamber stop codon is achieved by introducing an amber suppressortRNA/synthetase pair orthogonal to the endogenous tRNA complement intocells. Methanosarcina is a methane producing archaea with the unusualcapability of suppressing the stop codon (specifically the amber codon). Bysuppressing the amber codon Methanosarcina facilitate the incorporation of thenon-canonical amino acid pyrrolysine (pyl). The suppressing mechanismoriginates from a evolutionary unique Pyrrolysyl-tRNA synthetase (PylRS) and itsmatching tRNApyl. The PylRS has been further evolved and modified to allowincorporation of a wide range of UAAs. Amber suppression is today used tocontrol and study protein function in living cells. By making a series of wellcontrolledexperiments with HEK293T cells we aimed to develop this techniqueinto a robust and general tool for mammalian cell biology. Specifically we weretesting the incorporation of the unnatural amino acid bicyclononyne (BCN) by aset of known PylRS mutants. Our results suggest the mutant aaRS PylRS “AF” isthe most robust and efficient synthetase for BCN. We have improved ambersuppression by determining which factors leads to a more efficient method andsimultaneously decreasing the cost of the method.

Place, publisher, year, edition, pages
2016.
National Category
Other Biological Topics
Identifiers
URN: urn:nbn:se:kth:diva-190765OAI: oai:DiVA.org:kth-190765DiVA, id: diva2:1038973
Supervisors
Examiners
Available from: 2017-09-11 Created: 2016-08-15 Last updated: 2017-09-11Bibliographically approved

Open Access in DiVA

fulltext(7270 kB)70 downloads
File information
File name FULLTEXT02.pdfFile size 7270 kBChecksum SHA-512
a8201501adec1f060dcb9cf54906ef389bf79f946b06ca7cc28b418ecdbaeefd175354cb24b1fe07e6fb1f04b1644c3cb518a507103c405432baad0aeb673b21
Type fulltextMimetype application/pdf

By organisation
School of Biotechnology (BIO)
Other Biological Topics

Search outside of DiVA

GoogleGoogle Scholar
Total: 70 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

urn-nbn

Altmetric score

urn-nbn
Total: 410 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • harvard1
  • ieee
  • modern-language-association-8th-edition
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf