Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation
Abstract
Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate‐based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase‐coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell‐free protein synthesis activity. The resulting tRNA‐depleted lysate is compatible with in vitro‐transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real‐time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401–1407, 2017
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Jun 21, 2017
- Source ID
- 10.1002/btpr.2511
Entities
People
- Amin S M Salehi
- Bradley C. Bundy
- Brent L. Nielsen
- Christina Muhlestein
- Jeremy M. Hunt
- Joann Diray‐arce
- Mark T. Smith
- Song‐min Schinn
Organizations
- Brigham Young University
- Defense Advanced Research Projects Agency
- National Science Foundation