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Letter
Rapid and inexpensive evaluation of nonstandard amino acid incorporation in Escherichia coli Jordan W Monk, Sean P. Leonard, Colin W. Brown, Michael J. Hammerling, Catherine Mortensen, Alejandro E. Gutierrez, Nathan Y. Shin, Ella Watkins, Dennis M. Mishler, and Jeffrey E Barrick ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00192 • Publication Date (Web): 20 Sep 2016 Downloaded from http://pubs.acs.org on September 23, 2016
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Rapid and inexpensive evaluation of nonstandard
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amino acid incorporation in Escherichia coli
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Jordan W. Monk, Sean P. Leonard, Colin W. Brown, Michael J. Hammerling, Catherine
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Mortensen, Alejandro E. Gutierrez, Nathan Y. Shin, Ella Watkins, Dennis M. Mishler*, Jeffrey
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E. Barrick*
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Center for Systems and Synthetic Biology, Department of Molecular Biosciences, The
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University of Texas at Austin, Austin, TX 78712
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ABSTRACT
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By introducing engineered tRNA and aminoacyl-tRNA synthetase pairs into an organism, its
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genetic code can be expanded to incorporate nonstandard amino acids (nsAAs). The performance
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of these orthogonal translation systems (OTSs) varies greatly, however, with respect to the
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efficiency and accuracy of decoding a reassigned codon as the nsAA. To enable rapid and
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systematic comparisons of these critical parameters, we developed a toolkit for characterizing
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any Escherichia coli OTS that reassigns the amber stop codon (TAG). It assesses OTS
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performance by comparing how the fluorescence of strains carrying plasmids encoding a fused
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RFP-GFP reading frame, either with or without an intervening TAG codon, depends on the
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presence of the nsAA. We used this kit to (1) examine nsAA incorporation by seven different
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OTSs, (2) optimize nsAA concentration in growth media, (3) define the polyspecificity of an
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OTS, and (4) characterize evolved variants of amberless E. coli with improved growth rates.
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Keywords: expanded genetic code, noncanonical amino acid, unnatural amino acid, amber
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suppressor tRNA, nsAA measurement kit
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INTRODUCTION
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One goal of synthetic biology is to improve upon the limited chemical alphabets utilized by
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living organisms.1 The development of orthogonal translation systems (OTSs) has enabled a
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variety of nonstandard amino acids (nsAAs) to be incorporated into proteins in vivo.2,3 Many of
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these nsAAs can be used to create cells with novel capabilities. For example, some contain
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functional groups that enable halogen bonds4,5 or disulfide bonds6 with unusual properties
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compared to the interactions that normally stabilize protein structures. Other nsAAs can be used
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for bioorthogonal click chemistry,7 photocrosslinking,8 or photocleavage9 to further diversify the
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chemistry of cells and to exert precise spatiotemporal control of protein function.
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Artificially expanding the genetic code is typically accomplished by expressing a new tRNA
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and aminoacyl-tRNA synthetase (aaRS) pair in a host cell.2,3 In this setup, the additional aaRS
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has been engineered to recognize the nsAA and charge it onto the new tRNA. In E. coli, this
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tRNA is usually mutated so that it decodes the amber codon (TAG) as the nsAA. Recently, the
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functionality of OTSs has been further improved by deleting the protein release factor that
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competes with nsAA incorporation at the amber codon,10 engineering a protein elongation factor
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to better accommodate the OTS tRNA,11 and new methods for OTS directed evolution.12,13
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The performance of an OTS can be defined in terms of two main parameters, fidelity and
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efficiency.3,14,15 Fidelity refers to the accuracy with which the reassigned codon is read by the
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ribosome as the nsAA versus misread as any other amino acid. A lower fidelity OTS will result
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in proteins with mixture of the nsAA and one or more other amino acids at each TAG codon.
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Efficiency reflects how quickly the ribosome is able to read through the reassigned codon. A less
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efficient nsAA-incorporation system will result in a lower yield of output protein. The fidelity
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and efficiency of an OTS depend on many factors, including the rate of import of the nsAA into
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the host cell, the specificity of tRNA charging by the novel aaRS, and how effectively the new
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tRNA binds to and is translocated by the ribosome when it encounters the reassigned codon.14
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While these performance metrics are both derived from the biochemical properties of a particular
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orthogonal tRNA/aaRS pair, they are also somewhat independent, such that an OTS with low
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translational efficiency may still have very high fidelity, for example.
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OTS performance is typically assessed by expressing GFP or another reporter protein from
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an open reading frame containing one or more copies of the reassigned codon.13,16 Efficiency is
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judged by comparing the yield of this full-length reporter protein—monitored via a band on a
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gel, fluorescence, or enzyme activity—to a control gene with no copies of the reassigned codon.
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Fidelity is assessed by performing mass spectrometry on purified protein samples or by
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measuring protein yield from the recoded gene in the absence of the nsAA. While the first study
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creating a new OTS for a given nsAA typically characterizes the performance of several closely
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related aaRS and/or tRNA variants, OTSs derived from different studies or for incorporating
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different nsAAs are rarely compared in the same host cells under uniform growth conditions.
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Furthermore, there is no easy way for non-experts to cheaply and rapidly perform these
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measurements to ensure that an OTS is functioning in their hands or to screen many growth
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conditions to optimize OTS performance. Therefore, we designed and tested a genetic toolkit
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that can be used to assess the function of an OTS strictly from fluorescent read-out.
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RESULTS AND DISCUSSION
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Assessing orthogonal translation system performance
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Our method uses two reporter plasmids to measure the fidelity and efficiency of in vivo nsAA
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incorporation in E. coli (Fig. 1a). Each plasmid encodes mRFP1 and sfGFP fused into a single
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reading frame via a flexible peptide linker. In the control plasmid, pRYG, the DNA sequence
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encoding the linker contains a TAC tyrosine codon. The assay plasmid, pRXG, is identical
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except for a single point mutation that converts the TAC into a TAG amber codon to direct
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incorporation of the nsAA by the OTS. In this configuration, GFP signals readthrough of the
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focal TAC/TAG codon, and RFP serves as an internal control for any variance in overall protein
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production. Each version of the mRFP1-sfGFP fusion is terminated by a TAA stop codon after a
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C-terminal 6×His tag that can be used to purify the protein for mass spectrometry.
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To use the nsAA measurement kit, the control (pRYG) and assay (pRXG) plasmids are
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transformed separately into an E. coli strain containing an orthogonal translation system that
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recodes the amber codon to a nonstandard amino acid. In this study, each OTS consisted of an
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aaRS-tRNA pair encoded on plasmid pRF0G (Fig. 1b), and we tested OTS function in the
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genomically recoded "amberless" E. coli strain C321.∆A.exp.10 Performance of an OTS is
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evaluated by measuring RFP and GFP fluorescence (normalized to OD600) under four
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conditions: (1) assay plasmid with nsAA, (2) assay plasmid without nsAA, (3) control plasmid
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with nsAA, and (4) control plasmid without nsAA (Fig. 1c).
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The pRYG plasmid is a control for the relative GFP/RFP fluorescence signal expected for the
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entire fusion protein when there is efficient readthrough as a standard amino acid at this linker
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position. Therefore, the relative readthrough efficiency (RRE) of the TAG codon is the GFP/RFP
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fluorescence ratio for the pRXG assay plasmid divided by the GFP/RFP fluorescence ratio for
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the pRYG control plasmid (Fig. 1d). For an efficient aaRS-tRNA pair, the RRE when the nsAA
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is present in the media should approach or surpass a value of one, as this metric reflects how well
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the TAG amber codon is translated compared to the TAC tyrosine codon.
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RRE values do not contain any information about what amino acid is incorporated at the
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TAG codon, only how efficiently this codon is translated. The fidelity of an OTS is evaluated by
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comparing the RRE values obtained with and without nsAA present. Specifically, an (in)fidelity
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metric, the maximum misincorporation frequency (MMF), is calculated by dividing the RRE
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when nsAA is not added to the growth media by the RRE when nsAA is present (Fig. 1d).
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An ideal OTS has an MMF value of zero, reflecting that no GFP is produced unless the nsAA
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is present. Note, however, that MMF is a very strict measure of fidelity. Some engineered aaRS-
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tRNA pairs are known to incorporate mostly the nsAA when it is provided at a sufficiently high
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concentration, but to nonspecifically charge the tRNA with a standard amino acid instead when it
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is more abundant.12,15 Similarly, in the amberless E. coli host used in our experiments, translation
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is able to proceed to some extent through a single TAG codon, even when no cognate tRNA
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from an OTS is present.10 Presumably, this readthrough occurs because there is no other fate for
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ribosomes stalled on an amber codon except mistranslation by a noncognate tRNA or very slow
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dissociation, because release factor 1 is deleted in this strain. Thus, MMF should be interpreted
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as an upper bound on how often the TAG codon is incorrectly decoded as a standard amino acid.
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Comparing orthogonal translation systems
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We first used the kit to evaluate how well different nsAAs (Fig. 2) were incorporated into
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proteins by a panel of different OTS systems (Table 1), most derived from the
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Methanocaldococcus jannaschii tyrosine aaRS-tRNA pair. Time courses of OD600, RFP, and
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GFP signals were collected during growth of cells with each OTS and kit plasmid in LB media.
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For test conditions with nsAA present, it was added a concentration of 250 µM. We initially
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validated these measurements by examining the 3-iodotyrosine (IodoY) system, which we have
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found to have high fidelity and efficiency in previous studies.17,18 For the IodoY system, there is
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a large increase in GFP fluorescence for the pRXG assay plasmid when 3-iodotyrosine is present
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versus when it is absent in mid-log phase (Fig. 3a), as is expected for a system with high fidelity.
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The GFP signal also reaches similar levels for pRXG and the pRYG control plasmid when nsAA
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is present, which indicates 3-iodotyrosine is incorporated at the TAG codon with a similar
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efficiency to that of tyrosine at TAC. After cells have reached a saturating OD600, the specificity
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of the system appears to go down and GFP signal from the assay plasmid eventually increases in
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the condition lacking nsAA, which may indicate that misincorporation begins to occur at TAG
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during stationary phase.
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The time-course data collected for IodoY and three other tRNA-aaRS systems are
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representative of the variation in OTS performance that we observed. The AminoY OTS
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efficiently reads through the TAG codon (yielding GFP signal), whether or not the nsAA 3-
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aminotyrosine is provided (Fig. 3b). Thus, while it efficiently translates the amber codon, it has
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low fidelity and is expected to often decode TAG as amino acids other than the nsAA under our
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test conditions. NitroY exemplifies a third case (Fig. 3c). For this OTS, the signal of GFP from
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the assay plasmid is greater in the presence of nsAA than when it is not provided, but it does not
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reach anywhere near the level of control plasmid. Thus, the NitroY OTS accurately decodes
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TAG as 3-nitrotyrosine but with a very low translational efficiency. Finally, the function of the
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AzF system is very similar to that of the IodoY system (high efficiency and fidelity), except the
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fidelity of this OTS appears to remain high even after cells transition into stationary phase.
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To succinctly compare OTS performance, we present the rest of our measurements as the
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average RRE and MMF values over a time window when the cultures were in mid-log phase
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(Fig. 4a). We tested the Tyr OTS, which most of these nsAA systems are derived from, as an
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additional positive control. Like the IodoY system, it decodes TAG as efficiently as the TAC
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codon is translated as tyrosine by the native aaRS and tRNA pair (RRE value of ~1.0). We found
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that two of the three remaining OTSs for incorporating nsAAs functioned well, while one did
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not. The ONBY and 5HTP systems exhibit intermediate RRE and MMF values. Both have very
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low readthrough efficiencies for TAG when the nsAA is present, but they appear to maintain
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significantly better accuracy for nsAA incorporation than the AminoY system. Finally, the CNF
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system exhibits very low fidelity, much like the AminoY OTS, under our assay conditions.
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To better understand the relationship between the maximum misincorporation frequency (MMF) value measured by our kit and OTS fidelity, we employed protein mass spectrometry.
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Samples of the mRFP1-sfGFP fusion protein expressed in the presence of nsAA from the assay
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plasmid were purified from mid-log phase cultures of cells containing either the Tyr, IodoY,
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AminoY, NitroY, or CNF aaRS-tRNA systems. In comparison to the Tyr OTS control (Fig. S1),
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the mass peaks found in the IodoY (Fig. S2) and NitroY (Fig. S3) systems indicated that >95%
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of full-length proteins incorporated nsAA at the amber codon. This result for IodoY stresses how
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MMF establishes only a maximal limit on OTS infidelity (in this case,
