IP Library › Granted Patent US 9,834,775
Granted Patent B2
US 9,834,775 · App. 15/025,406 · Granted Dec 5, 2017

Recombinant cells and organisms having persistent nonstandard amino acid dependence and methods of making them

Inventors: George M. Church (Brookline, MA); Daniel J. Mandell (Brookline, MA); Marc J. Lajoie (Cambridge, MA)
Assignee: President and Fellows of Harvard College
C12N15/65C12N1/36C12N15/52C12N2800/22C12N2840/102
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Quick Facts
Patent No.
US 9,834,775
App. No.
15/025,406
Granted
Dec 5, 2017
Kind
B2
Abstract

Recombinant cells and recombinant organisms persistently expressing nonstandard amino acids (NSAAs) are provided. Methods of making recombinant cells and recombinant organisms dependent on persistently expressing NSAAs for survival are also provided. These methods may be used to make safe recombinant cells and recombinant organisms and/or to provide a selective pressure to maintain one or more reassigned codon functions in recombinant cells and recombinant organisms.

Claims (8)

1. A genetically modified Escherichia coli ( E. coli ) cell comprising:

an essential polypeptide encoded by an essential nucleic acid sequence, wherein the essential polypeptide comprises a recoded adenylate kinase (adk) comprising I4A, L6V, V103A, L178bipA, Y182V, and T191I substitutions; a recoded alanyl-tRNA synthetase (alaS) comprising M1bipA, F90A, F293A, L338bipA, M342A, and L349P substitutions; a recoded methionyl-tRNA synthetase (metG) comprising M485A, F502G, and L503bipA substitutions; a recoded phosphoglycerate kinase (pgk) comprising V185A, I187A, I211G, and L297bipA substitutions; or a recoded tyrosol-tRNA synthetase (tyrS) comprising F706A, W778F, T787V, F823G, L907bipA, and V919A substitutions, wherein bipA is L-4,4′-bisphenylalanine.

2. The cell of claim 1 , comprising two or more essential polypeptides encoded by two or more essential nucleic acid sequences.

3. The cell of claim 1 wherein the essential polypeptide comprises the recoded adk comprising I4A, L6V, V103A, L178bipA, Y182V, and T191I substitutions.

4. The cell of claim 1 wherein the essential polypeptide comprises the recoded alaS comprising M1bipA, F90A, F293A, L338bipA, M342A, and L349P substitutions.

5. The cell of claim 1 wherein the essential polypeptide comprises the recoded metG comprising M485A, F502G, and L503bipA substitutions.

6. The cell of claim 1 wherein the essential polypeptide comprises the recoded pgk comprising V185A, I187A, I211G, and L297bipA substitutions.

7. The cell of claim 1 wherein the essential polypeptide comprises the recoded tyrS comprising F706A, W778F, T787V, F823G, L907bipA, and V919A substitutions.

Assignments (3)
CONFIRMATORY LICENSE Recorded Feb 12, 2025
From: HARVARD UNIVERSITY
To: NATIONAL SCIENCE FOUNDATION
Reel/Frame 070191/0443 →
ASSIGNMENT OF ASSIGNOR'S INTEREST Recorded Feb 12, 2018
From: CHURCH, GEORGE M.; LAJOIE, MARC J.; MANDELL, DANIEL J.
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 044894/0197 →
CONFIRMATORY LICENSE Recorded Mar 31, 2016
From: HARVARD UNIVERSITY
To: NATIONAL SCIENCE FOUNDATION
Reel/Frame 038312/0198 →
Continuity (2)
Provisional Application 61883413 · Sep 27, 2013
Related Publication 20160230176A1 · Aug 11, 2016