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Expanding the Genetic Code:
Measurement Kit for the incorporation of non-canonical amino acids
Alejandro Gutierrez, Jordan Monk, Catherine Mortensen, Nathan Shin, Ella Watkins.
Advisors: Dennis Mishler, Ph.D. and Professor Jeffrey Barrick
University of Texas at Austin, iGEM 2014 team: Austin_Texas
Motivation and Background
In recent years, the ability to expand the genetic code has been
made possible by re-coding the amber stop codon, UAG, via the
use of modified tRNA synthetase/tRNA pairs. The modified tRNA
synthetase charges the modified tRNA with a non-canonical amino
acid (ncAA), an amino acid that is not one of the 20 amino acids
naturally encoded. Many different ncAA synthetase/tRNA pairs
have been created, which allows for the introduction of novel
functions into proteins. While numerous ncAA synthetase/tRNA
pairs have been engineered, many are poorly characterized and
may not specifically charge or not efficiently
incorporate their ncAA.
Measurement Kit
Measuring Fluorescence
The 4-Azidophenylalanine (AzF)
pair shows high fidelity.
The 3-Aminotyrosine (AminoY)
pair shows poor fidelity.
Incorporation Value (I)
•  A single value that indicates the
efficiency and fidelity of a
synthetase/tRNA pair.
•  The greater the value, the better the
pair.
•  Controls (pFRYC) should be ~1.
•  Experimentals (pFRY) should be
greater than 1.
•  A value less than 1 indicates low
efficiency and/or toxicity.
•  4 of the pairs tested are good!
•  3 of the pairs tested are bad.
•  We created the Expanded Genetic Code Measurement Kit.
Our kit can be used to quantify the fidelity and efficiency of
any ncAA synthetase/tRNA pair.
•  We also used one of these ncAAs to create a system of lightactivated protein expression through Photocaged T7 RNAP.
Methods
The Expansion Pack Test Kit Plasmids
GF
P
Lac
O
pFRYC
pFRY
La
La
Ge
nt R
Ka
nR
Control Strain
Experimental Strain
GF
P
P
RF
cI
pStG
Lac
O
Synthe
tas
e
P
RF
cI
tRNA
Ka
nR
•  pStG - tRNA synthetase/tRNA pair.
•  pFRYC - RFP-GFP reporter protein
connected by a linker that does
NOT contain an amber codon.
•  pFRY - RFP-GFP reporter protein
connected by a linker that contains
an amber codon (star).
•  The control strain contains
pFRYC and pStG.
•  The RFP-GFP reporter should be
produced in all conditions.
ONBY: Photocaged T7 RNAP
ortho-nitrobenzyl Tyrosine (ONBY)
•  ONBY can be used to photocage
proteins.
•  The ONB group can be cleaved off with
365 nm light.
Tyrosine
ortho-nitrobenzyl Tyrosine (ONBY)
Photocaged T7 RNA Polymerase (RNAP)
365 nm Light
•  The experimental strain
contains pFRY and pStG.
•  Without ncAA, only RFP should
be produced.
•  With ncAA, the RFP-GFP
reporter should be produced.
•  Graphs show the amount of GFP
fluorescence measured relative to
RFP fluorescence.
•  Control GFP:RFP (pFRYC) is
independent of ncAA.
•  Experimental GFP:RFP (pFRY) is
dependent on the quality of the
tRNA synthetase/tRNA pair and the
presence of ncAA.
Caged T7 RNAP
mRNA
Translation
DNA
Active T7 RNAP
Protein
These experiments are conducted in amberless E. coli, which have no amber codons in their genomes (Lajoie 2013). •  ONBY can be incorporated at tyrosine
639 in T7 RNAP to prevent activity until
decaged (Chou 2010).
•  Can be paired with any gene under the
control of a T7 promoter.
•  Giving us spatiotemporal control over
the transcription of that reporter via
ONBY incorporation in T7 RNAP.
Exposure *me Achievements
•  The 2014 UT Austin iGEM successfully created the Expanded Genetic Code
Measurement Kit, including reporter plasmids in amberless E. coli.
•  Our system is inexpensive, portable, easy to use, and can quickly characterize
numerous ncAA synthetase/tRNA pairs.
•  We characterized seven ncAA synthetase/tRNA pairs to demonstrate the quality
of the system.
•  We successfully recreated a Photocaged T7 RNAP, demonstrating
spatiotemporal control over reporter gene transcription.
•  We submitted BioBricks for two of the functional tRNA synthetase/tRNA pairs
•  Our Measurement Kit is now available to anyone who requests it.
Temporal control of GFP expression
using light-activated T7 RNAP.
Spatial control of GFP expression
using light-activated T7 RNAP.
Special thanks to: Barrick Lab, Razan Alnahhas, Dr. Colin Brown, Michael J. Hammerling, Sean Leonard,
and Ashley Kessel
References: Chou, C., Young, D. D. and Deiters, A. (2010). Lajoie, M.J., Rovner, A.J., Goodman, D.B., et al. (2013). FUNDING FROM: