This document discusses technologies for genome engineering including MAGE and CAGE. MAGE allows for introducing multiple genetic changes at multiple locations while CAGE uses bacterial conjugation to produce extensively modified genomes. The goal is to achieve virus resistance, genetic isolation, and stable expansion of the genetic code. This involves artificially modifying the genetic code by reassigning codons to encode non-standard amino acids. Challenges include overcoming the conservation and complexity of the genetic code as well as biochemical constraints within the cell.

1. EXPANDEDGENETICCODE
engineering

2. GENOME ENGINEERING TECHNOLOGIES
MAGE & CAGE

3. MAGE
• Multiplex automated genome
engineering
• Introduction of multiple
changes
• Multiple locations

4. CAGE
• conjugative assembly genome
engineering
• bacterial conjugation
• production of extensively modified
genomes

5. To achieve…
• Virus
resistance
• genetic
isolation
• stable
expansion
searchable genome annotation
databases
sequence manipulation tools
synthetic circuit design tools
 sequencing analysis tools

6. • artificially modified genetic code
• Specific codons have been re-allocated

7. • non-standard amino acid to
encode
• an unused codon
• a tRNA
• a tRNA synthetase

8. ENGINEERING EXPANDED
GENETICCODES

9. • In vitro flexibility
• Incorporation of
nsAA
Formation of un-
natural backbone
for translation
Best way

10. CODON SUPPRESSION
In Vivo…
• Inexpensive, simple
translations
Risk
• Deleterious
metabolic system
Evolution of
tolerance
more than
167 nsAAs
medicine and
bioremediation

11. CODON REASSIGNMENT
Hijacking…
• Re-assigning a codon
function
E.Coli
• Amber codon (UAG)
• tRNATyr / tyrosyl-
tRNA synthetase

12. General Strategy
• Identification
• Abolishing the natural functions
• Introduction of new functions

13. CODON CREATION
Beyond Re-purposing…
• Addition of a
base pair Immediate implementation
• High fidelity
• Efficient proof
reading

14. CHALLENGES
• “refractory” to
change
• Conservation factors
• Biological complexity
(580,070 base pairs)
• biochemical
constraints
compensati
ons

15. BARRIERS TO CHANGE
GENETIC CODE

16. BIOCHEMICAL BARRIERS
Process:
Amino
acids
• aminoac
yl-tRNA
synthet
ases
tRNA
• Elongati
on
factor
Ribosome
• Active
site

17. GENETIC BARRIERS
single nucleotide
variants…
…genome
architecture.

18. GENOME ENGINEERING BARRIERS
• Prediction of
changes
• draft genomes

19. Abolishing codon functions
Addition of nsAA
Introduction of unnatural
functions
CHALLENGE??