Evolving Orthogonal Suppressor tRNAs To Incorporate Modified

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Evolving orthogonal suppressor tRNAs to incorporate modified amino acids Andre Maranhao, and Andrew D. Ellington ACS Synth. Biol., Just Accepted Manuscript • DOI: 10.1021/acssynbio.6b00145 • Publication Date (Web): 07 Sep 2016 Downloaded from http://pubs.acs.org on September 8, 2016

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ACS Synthetic Biology

Title: Evolving orthogonal suppressor tRNAs to incorporate modified amino acids Authors: Andre C. Maranhao1, Andrew D. Ellington1* 1. Center for Systems and Synthetic Biology, University of Texas at Austin, Austin,

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Texas 78712 Abstract

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There have been considerable advancements in the incorporation of non-canonical

8

amino acids (ncAA) into proteins over the last two decades. The most widely used method

9

for site-specific incorporation of non-canonical amino acids, amber stop codon suppression,

10

typically employs an orthogonal translation system (OTS) consisting of a heterologous

11

aminoacyl-tRNA synthetase:tRNA pair that can potentially expand an organism’s genetic

12

code. However, the orthogonal machinery sometimes imposes fitness costs on an organism,

13

in part due to mischarging and a lack of specificity. Using compartmentalized partnered

14

replication (CPR) and a newly developed pheS negative selection, we evolved several new

15

orthogonal Methanocaldococcus jannaschii (Mj) tRNA variants tRNAs with increased amber

16

suppression activity, but that also showed up to three-fold reduction in promiscuous

17

aminoacylation by endogenous aminoacyl-tRNA synthetases (aaRSs).

18

orthogonality of these variants greatly reduced organismal fitness costs associated in part

19

due to tRNA mischarging. Using these methods, we were also able to evolve tRNAs that

20

supported the specific incorporation of 3-halo-tyrosines (3-Cl-Y, 3-Br-Y, and 3-I-Y) in E. coli.

The increased

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Keywords: non-canonical amino acids, orthogonal translation systems, tRNA, aminoacyl-tRNA

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synthetase

24 25

Introduction

ACS Paragon Plus Environment


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Over the last two decades, researchers have attempted to create proteins with new

2

functionalities through the incorporation of non-canonical amino acids. Expanding the

3

genetic code has in turn typically been achieved through the introduction of orthogonal

4

aaRS:tRNA pairs, especially pairs that rely on suppressor tRNAs. The archaeal M. jannaschii

5

(Mj) tyrosyl-tRNA syntethase:tRNA pair has proven particularly tractable for the

6

introduction of non-canonical amino acids in E. coli, in that both the synthetase and the

7

tRNA are largely orthogonal to the other aaRSs and their cognate tRNAs1-4. However, most

8

experiments to date have attempted to introduce only a single non-canonical amino acid

9

into a protein, and have not attempted multiple incorporations or proteome-wide

10

incorporation. This is in part because there are concomitant fitness costs to altering the

11

genetic code. In particular, it is known that inaccurate translation, such as frameshifting

12

and false termination, can lead to fitness costs5. In addition, recent work has demonstrated

13

that E. coli strains containing orthogonal charging machinery will quickly delete the newly

14

introduced MjYRS6. We hypothesize that this may be due in part to the lack of charging

15

fidelity: the wrong amino acid can go on tyrosine tRNA, or the wrong tRNA can be charged

16

with tyrosine.

17

In other work, it was shown that promiscuous interactions between a p-

18

acetylphenylalanine incorporating variant of MjYRS and E. coli prolyl-tRNAs could be

19

remedied through over-expression of the endogenous E. coli prolyl-tRNA synthetase7.

20

Similarly, promiscuous interactions between endogenous E. coli tRNAs and an introduced

21

heterologous aaRS can be mitigated by making that heterologous synthetase inducible as

22

well as by increasing the expression level and hence the concentration of the paired

23

heterologous tRNA. However, doing so shifts the burden for orthogonal behavior unto the

24

heterologous tRNA.


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There are various routes that can be pursued to improve the ability of cells to

2

incorporate non-canonical amino acids and to increase the fitness of the resulting expanded

3

genetic codes. Isaacs and co-workers have developed an E. coli strain that completely lacks

4

amber codons8, and this should limit fitness effects that arise from amber suppression.

5

However, the effort required for the development of this strain was extraordinary, and the

6

use of wholescale genome engineering to alter the genetic code is not yet widely available.

7

The use of directed evolution methods to improve the efficiency and reduce the promiscuity

8

of charging complements rational design approaches9.

9

Therefore, we have attempted to utilize a powerful directed evolution method,

10

compartmentalized partnered replication (CPR)10 to optimize the M. jannaschii tyrosine

11

suppressor tRNA for efficient and specific suppression. To ensure improved orthogonal

12

behavior, we implemented a new negative selection scheme in place of the widely used

13

barnase2,11. We instead adapted a mutant E. coli phenylalanyl-tRNA synthetase (PheS-

14

A294G) with relaxed substrate specificity to create a robust and tunable negative selection

15

method via the introduction of the toxic p-Cl-phenylalanine12. This selection functions to

16

select against mischarging of MjY-tRNA variants by the host aaRS machinery.

17

By using CPR with negative selection, we have generated a new series of tRNAs with

18

enhanced amber suppression activity for not only tyrosine, but also for three non-canonical

19

amino acids. These tRNAs show up to three-fold reduction in promiscuous aminoacylation

20

by endogenous aaRSs, and rescued reduced cell growth observed in the parental M.

21

jannaschii tyrosine suppressor tRNA.

22 23

Results and Discussion

24

Selection Strategy



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1

A technique known as compartmentalized partnered replication (CPR) has

2

previously been used for the directed evolution of functional and specific tRNAs10. A

3

general schematic for CPR can be found in Figure 1; in short, tRNA libraries are expressed

4

in individual E. coli cells, which are in turn emulsified into individual droplets. Functional

5

suppressor tRNAs lead to the production of Taq polymerase, and emulsion PCR in the

6

presence of tRNA-specific primers in turn leads to the amplification of those tRNA variants

7

responsible for suppression and Taq polymerase production.

8

CPR mock selections were first conducted to optimize the protocol for amber

9

suppression with the Methanocaldococcus jannaschii (Mj) tyrosine tRNA in combination

10

with Mj tyrosyl-tRNA synthetase (MjYRS).

11

tRNAoptCUA3, an efficient suppressor that has previously been partially optimized by directed

12

evolution. The tRNAoptCUA was placed under the control of the lpp promoter, which has

13

previously been used in successful suppression experiments1. In order to ensure that

14

selected tRNAs would be efficient suppressors, four amber codons were simultaneously

15

introduced into Taq polymerase. The T7-driven Taq polymerase and all aaRS genes were

16

under the control of the tacI promoter and were induced by IPTG.

Mock selections were carried out with

17

Initial ranging selections were carried out in which E. coli BL21(DE3) cells

18

expressing wild MjYRS were competed with cells containing an E. coli seryl-tRNA synthetase

19

(EcSRS), which should not aminoacylate tRNAoptCUA. Cells bearing the EcSRS gene should not

20

suppress amber codons, and thus any proteins or functions that are produced (such as DNA

21

amplification via

22

aminoacylation of tRNAoptCUA by endogenous E. coli synthetases. We determined that cells

23

induced with 0.4 mM IPTG for three hours prior to emulsification provided the optimal

24

conditions for ensuring emulsion PCR amplification by cells expressing MjYRS, while

25

eliminating similar amplification by cells expressing EcSRS.

Taq polymerase) represent background


due

to

promiscuous

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1


The level of enrichment during selection was determined by competitions with a

2

disrupted, inactive form of MjYRS that contained an introduced NotI site.

3

suppression and amplification, we recovered emulsion PCR products from different ratios

4

of cells containing functional MjYRS to cells containing inactive MjYRS. After three rounds

5

of mock selections, equal amounts of PCR product from each round of CPR were digested

6

with NotI and the digestion products were analyzed by gel electrophoresis. From these gels,

7

it was estimated that about 103-fold enrichment of active over inactive MjYRS could be

8

expected for each round of CPR (Figure S1).

Following

9 10

Development of a negative selection method

11

To date, we have primarily employed CPR for positive selection. In order to ensure

12

orthogonal behavior with respect to endogenous E. coli synthetases, there is a need for

13

negative selection of suppressor tRNA libraries, especially a negative selection whose

14

stringency can be tuned. To this end, we have adapted a negative selection based on the

15

mutant E. coli synthetase PheS

16

specificity that allows aminoacylation of E coli tRNAPhe with p-Cl-phenylalanine (p-Cl-Phe),

17

which upon incorporation into the proteome leads to cell death.

12,

a variant of PheS with relaxed substrate binding

18

By having tRNA variants suppress the expression of mutant PheS, rather than Taq

19

polymerase, a negative selection could be imposed, and the selection could be tuned by

20

varying the concentration of p-Cl-Phe and the duration of growth in media containing p-Cl-

21

Phe. We implemented this negative selection technique via ACP-(PheS-A294G) fusion

22

protein possessing a flexible linker with two amber codons. Passage in liquid culture

23

containing p-Cl-Phe should cull the library of promiscuous tRNA variants expressed in the

24

absence of the cognate Mj synthetase.

25



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1

Selection for improved tyrosine suppressor tRNAs

2

The previously engineered tRNAoptCUA suppressor was also used as a starting point

3

for library design. The sequence and structure of tRNAoptCUA (Figure 2A) were compared

4

with the sequences and secondary structures of other, non-suppressor E. coli tRNAs in

5

order to determine the likely features that might lead to mischarging. When studying their

6

secondary structures, similarities were observed between tRNAoptCUA and the T-arms and

7

anticodon stems of tRNAoptCUA and EctRNAserU.

8

generated two first-generation libraries that randomized residues in the anticodon region

9

(Ac-Lib) and T-arm (T-Lib) (Figure 2A), a strategy that had previously proven successful

10

for the isolation of other improved tRNA variants10. Each of these libraries contained 4.2 x

11

106 variants, a convenient number for screening via emulsion-based methods. The

12

sequences for all relevant tRNA variants can be found in Table S1.

Using tRNAoptCUA as the template, we

13

Ac-Lib and T-Lib libraries were subjected to three rounds of CPR positive selection,

14

one round of pheS negative selection, and then a final round of CPR positive selection.

15

During the first two rounds of CPR, selections were carried out under the same conditions

16

as the mock selections described above. However, for the third and fifth rounds of CPR,

17

tRNA library pools were placed under the control of the stronger E. coli leuQ tRNA promoter

18

and challenged with a Taq polymerase gene that contained six amber stop codons. We

19

hypothesized that selection under the control of a very strong tRNA promoter would lead to

20

greater competition with endogenous cellular tRNAs, and that any deleterious off-target

21

effects that occurred would help cull promiscuous, toxic variants from the pool during the

22

overnight growth step prior to selection.

23

Following selection, the tRNA pools were cloned and sequenced. The Ac-Lib had

24

collapsed to one anticodon stem sequence, while the T-lib displayed somewhat greater T-

25

arm sequence diversity.

Interestingly, all sequenced Ac-Lib and T-Lib tRNA variants


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(including the most active variants, Figure S2) possessed one or more mutations outside

2

the randomized library region (Figure S3). Some of these non-library mutations were in

3

either the tRNA promoter or in the flanking 5’ and 3’ tRNA processing sequences; because

4

these likely changed expression or processing, rather than intrinsic tRNA function, these

5

variants were excluded from further characterization.

6

Suppression activities for twenty individual tRNA variants with wild-type flanking

7

sequences were determined via GFP suppression efficiency assays. Suppression efficiency

8

was measured in the presence of a given evolved tRNA and its cognate synthetase, a GFP

9

reporter gene that contained three amber codons in place of three surface exposed

10

tyrosines (Y39, Y151, Y182) was used. GFP should only be made if the evolved tRNA is

11

efficiently charged by its synthetase. Suppression orthogonality was measured in the

12

presence of a given evolved tRNA but in the absence of the cognate synthetase, a GFP

13

reporter gene that contained only one amber at position Y39 was used. In this case, GFP

14

should only be made if there is significant mischarging of the tRNA by an endogenous aaRS.

15

The most active Ac-Lib variants proved to be Ac.02, Ac.10, and Ac.11, all of which

16

possessed the same anticodon stem sequence and additional mutations elsewhere in the

17

tRNA. Variant Ac.02 had two non-library mutations: G71A in the acceptor stem and A57G

18

in the T-loop. Variants Ac.10 and Ac.11 had an additional acceptor stem (C70U) and T-loop

19

(U54C) mutation, respectively.

20

mutations, C70U and G71A were removed from Ac.02 and Ac.10, but variants lacking these

21

mutations could not be successfully cloned under the leuQ promoter.

During initial attempts to study these non-library

22

The most active T-Lib variants were T.08 and T.12 contained different T-arm

23

sequences, but possessed the same C74U mutation at the 3’ end of the tRNA. This non-

24

library, T-arm mutation was excluded from further consideration as it is known to lead to

25

cleavage and re-addition of 3’ CCA, which adds another step to tRNA processing and



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1

effectively slows maturation13.

2

important for tRNA behavior.

The remaining non-library mutations were clearly

3

In order to create a tRNA variant with even greater efficiency and orthogonality, we

4

attempted to combine the Ac-Lib consensus anticodon stem sequence (and accompanying

5

non-library mutations C70U and G71A) with combinations of the selected T-arm variants

6

(T.08 and T.12) and with non-library T-loop mutations – A57G from variant Ac.02 and U54C

7

from variant Ac.10. Ten combinatorial variants were generated (Figure S4). AcT.07 and

8

AcT.09 lacked the U54C non-library mutation from the T-loop and could not be stably

9

cloned, again indicating the importance of these selected, spontaneous mutations for tRNA

10

function.

11

From the eight stably cloned combinatorial variants, only variant AcT.05 displayed

12

amber suppression efficiency and orthogonality levels comparable to those of the parental

13

Ac.Lib and T.Lib tRNA variants. Variant AcT.05 (Figure 2B) derives from combination of

14

the common anticodon stem sequence, the T-arm from variant T.08, and the C70U non-

15

library mutation from Ac.02.

16

suppression efficiency and orthogonality (Figure S5), was thus the best and most diverse

17

tRNA variant, and was adopted as a platform for the further evolution of highly orthogonal

18

tRNAs.

Variant AcT.05 had a favorable combination of high

19 20

Second-generation tyrosine suppressor tRNA selection

21

A new library based on AcT.05 (AcT5S; Figure S6) was generated that further

22

varied those positions where non-library mutations were found during the first set of

23

selections (Figure S3). This library was subjected to two rounds of CPR, followed by one

24

round of pheS negative selection, and then three more rounds of CPR. Library members had

25

to suppress Taq polymerase genes containing six and eight amber codons during the first


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1

and second rounds of CPR, respectively.

2

polymerase gene contained ten amber codons.

In the final three rounds of CPR, the Taq

3

Post-selection sequencing of the selected AcT5S library revealed almost no non-

4

library mutations, validating our strategy of varying these additional positions to further

5

improve functionality. However, no sequence convergence occurred in either library and

6

considerable diversity was still observed. Of the thirty six variants assessed, the most

7

efficient tRNA variants were re-assayed (Figure S7) and variant S7 was found to be the

8

most efficient and orthogonal. Variant S7 (Figure 2C) displayed slightly higher amber

9

suppression efficiency compared to both the parental tRNA, AcT.05 and to tRNAoptCUA

10

(Figure 2D). Variant S7 and AcT.05 display comparable levels of orthogonal behavior, and

11

are considerably more orthogonal than tRNAoptCUA (Figure 2E).

12

Variant S7 contains two new G:U wobble base pairs in its acceptor stem.

13

Interestingly, several other active variants from the AcT5S library pool (Figure S8) possess

14

at least one G:U wobble base pair in the acceptor stem, and three other AcT5S variants have

15

two; all of these occur at different positions. Wobble base pairs in tRNA stems have

16

previously been shown to contribute to recognition14,15, in part by modifying the structure

17

or flexibility of the stem. For example, the G3:U70 wobble in A. flugidus tRNAAla kinks the

18

tRNA and so properly orients the tRNA’s 3’-CCA with the paired synthetase’s active site16.

19

Such stem distortions and concomitant alterations in flexibility are common in tRNAs for

20

class I aminoacyl-tRNA synthetases17. In at least one case, the flexibility of the arginyl tRNA

21

from hamster plays a role in its binding to and recognition by its synthetase18. In previous

22

work from our laboratory, it was shown that removing a single wobble pair (U69C) from the

23

Saccharomyces cerevisiae tryptophanyl tRNA acceptor stem led to charging by E. coli

24

histidinyl tRNA element9. Changes to acceptor stem flexibility appear to play a significant

25

role in tRNA recognition by aaRSs, both cognate and non-cognate. Changes to the acceptor



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1

stem sequence are also known to impact EF-Tu binding

2

flexibility impacts not only charging but loading of tRNAs.

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19,20,

and it is possible that stem

3 4

Selecting for increased 3-halo-tyrosine incorporation activity

5

The tRNA variants S7 and tRNAoptCUA were also assayed for their ability to

6

incorporate the non-canonical amino acid 3-iodo-tyrosine21. In the presence of the non-

7

canonical amino acid and its cognate synthetase, variant S7 incorporated 3-iodo-tyrosine at

8

less than 10% the activity observed with both tRNAoptCUA and another previously selected

9

tRNA variant Nap1, a similar suppressor tRNA that was evolved to optimize EF-Tu binding

10

and has been successfully used to incorporate numerous non-canonical amino acids2. As all

11

the selections which ultimately led to S7 tRNA were carried out with tyrosine, we reasoned

12

that this may have led to over-optimization for tyrosine incorporation. This may have in

13

turn resulted in poor suppression efficiency with amino acid analogues, possibly because

14

balanced binding to EF-Tu by both a given tRNA and the charged amino acid are required

15

for efficient translation.20,22,23.

16

To test this hypothesis, we attempted to rescue the ability of S7 to incorporate non-

17

canonical amino acids by carrying out a selection with the MjYRS variant

18

specifically incorporates 3-halo-tyrosine analogs, and is known not to utilize tyrosine.

19

Library designs (S7-TA, Figure 3A) were focused on positions in the tRNA known to

20

interact with EF-Tu: positions 3, 6, 68, and 71 in the acceptor stem25, and positions 49-51

21

and 63-65 in the T-arm20. We and others have reasoned that simultaneous selection of the

22

acceptor stem and T-arm would be most likely to yield highly efficient tRNAs2,26. Recalling

23

that a number of different acceptor stems recovered from the AcT5S library contained G:U

24

base-pairs, positions 3, 6, 68, and 71 in the acceptor stem of the S7-TA library were

25

restricted to either C or U.


21,24

that

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1

Starting from the S7-TA library, two-round CPR selections were conducted with

2

three different 3-halo-tyrosines: 3-chloro-tyrosine (3-Cl-Y), 3-bromo-tyrosine (3-Br-Y), and

3

3-iodo-tyrosine (3-I-Y). Following an initial screen of thirty-six colonies, highly efficient

4

variants were screened by again examining the expression of a GFP gene that contained

5

three amber stop codons. Rescreening in triplicate led to the identification of the two most

6

active tRNA variants for each amino acid. Of the final six tRNA variants, one was the second

7

most active variant in both the 3-I-Y S7-TA library pool (C1) and the 3-Br-Y S7-TA library

8

pool (G8).

9

The final five 3-halo-tyrosine tRNA variants (E4, E2, C1/G8, E7, and G2; Figure 3B)

10

were assessed in suppression efficiency assays with 3-Cl-Y, 3-Br-Y, and 3-I-Y (Figure 3C).

11

These variants not only rescued the low efficiency of amber suppression with 3-halo

12

tyrosines originally observed with the parental S7 tRNA, but in some instances enhanced

13

amber suppression efficiency relative to previously evolved tRNAoptCUA and Nap1.

14

comparison to the parental S7 tRNA variant, all five 3-halo-tyrosine tRNA variants displayed

15

between 12- and 16-fold increases in suppression efficiency with 3-Br-Y and 3-I-Y while

16

increasing suppression efficiency with 3-Cl-Y by between 16- and 20-fold. In particular,

17

variant C1/G8 displayed over 14-fold increases in suppression efficiency with all 3-halo-

18

tyorinses relative to its parental S7 tRNA variant, and gains of 20% and 6% suppression

19

efficiency relative to tRNAoptCUA for 3-I-Y and 3-Br-Y incorporation.

In

20

In order to measure the orthogonality of suppression, the tRNAs were again assayed

21

for the ability to suppress a GFP gene bearing only one amber stop codon in the absence of

22

their cognate synthetase (Figure 3D). Interestingly, positions 3 and 71 universally fixed to

23

cytosine for the most active tRNA variants from this selection, again pointing to the

24

potential role of tRNA flexibility in recognition.

25

orthogonal behavior comparable to that of the parental tRNA variants AcT.05 and S7, while

Only variant C1/G8 demonstrated



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1

other selected tRNAs seem to lose orthogonality.

2

optimization for function with tyrosine result in reductions in mischarging, that

3

optimization for function with a non-canonical amino acid at least initially occur along

4

pathways that lead to interactions with non-cognate synthetases or incorrect proofreading

5

by EF-Tu.

These results suggest that while

6 7

Selected suppressor tRNAs improve organismal fitness

8

In order to examine how CPR-based selections for improved suppression efficiency

9

and orthogonality also impacted organismal fitness, we compared the most orthogonal

10

selected variant, S7, with the previously evolved tRNAoptCUA and Nap1. These tRNAs were all

11

expressed under the control of the leuQ tRNA promoter in the strains DH10B E. coli and the

12

“Amberless” E. coli C321.ΔA (Figure 4). Each growth curve represents triplicate averages

13

which were subsequently fitted to a modified logistic growth model27 that estimates

14

maximum specific growth rate and lag time. Table S2 presents these two parameters and

15

their respective errors for each growth assay. Cells expressing tRNAoptCUA or Nap1 grew

16

noticeably slower, while the newly selected tRNA variant S7 appeared to reverse this

17

growth defect in DH10B E. coli (Figure 4A). This improvement in fitness also appeared to

18

be the case for all of the descendants of S7, including the seven 3-halo-tyrosine

19

incorporating tRNA variants, which for the most part grew better relative to the previously

20

selected tRNAs tRNAoptCUA and Nap1 (Figure S9).

21

The expression of a tRNA without its paired synthetase of course does not reflect

22

normal expression conditions, and additional growth assays were conducted with

23

constructs expressing either the wild-type tyrosyl-tRNA synthetase (MjYRS) or a variant of

24

MjYRS that specifically incorporates 3-halo-tyrosines22 (Mj(3IY)RS), both driven by

25

induction of the tacI promoter. Assessment with Mj(3IY)RS in the absence of 3-halo-


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1

tyrosines affords the opportunity to study growth effects when a heterologous synthetase

2

can bind to a tRNA but cannot aminoacylate it. Each tRNA and synthetase pair was grown in

3

either 2xYT media with 2% glucose for catabolic repression of the paired synthetase, 2xYT

4

for leaky expression of the synthetase, or 2xYT media with 0.1 mM IPTG for induction of the

5

synthetase. These growth assays were again conducted using the three tRNA variants

6

(Nap1, tRNAoptCUA, and S7) in either DH10B E. coli (Figure 5) or “Amberless” E. coli C321.ΔA

7

(Figure 6). In DH10B E. coli, growth of the suppressor tRNA variant S7 paired with the

8

wild-type MjYRS is roughly equivalent to that of a pBR322 control given replicate error

9

(Figure 5, upper left). However, both the tRNAoptCUA and Nap1 variants demonstrated

10

markedly slower growth phenotypes.

11

tRNAs gave similar growth curves, irrespective of provenance (Figure 6). Overall these

12

findings suggest that fitness effects from orthogonal charging systems in large part result

13

from toxicities associated with amber suppression by promiscuously aminoacylated tRNAs.

14

This finding is echoed by the fact that S7 is rendered toxic to cells when its paired

15

synthetase is overexpressed (Figure 5, lower left).

In contrast, in the “Amberless” E. coli C321.ΔA, all

16 17

Conclusion

18

Transfer RNA sits at the crux of multiple biochemical processes required for

19

translation, and the directed evolution of tRNAs has therefore proven to be a powerful tool

20

for changing the translation apparatus. We have selected several Mj tyrosine suppressor

21

tRNA variants that have both improved suppression efficiencies and reduced mischarging

22

by endogenous aaRSs. We achieved improved orthogonality by incorporating a novel

23

liquid-based pheS negative selection. The accumulated changes in tRNA orthogonality also

24

led to decreased fitness costs to organisms that contain these tRNAs. The impact of tRNA

25

identity on fitness has only rarely been gauged; previous analysis has focused on the fitness



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Page 14 of 36

1

costs of codon reassignment rather than tRNA orthogonality28. We used the same selection

2

methods to further generate tRNA variants that could efficiently incorporate halo-tyrosines.

3

The sequence of any given tRNA can be seen as a balancing act for multiple contacts

4

with different partners, from synthetases to EF-Tu to the ribosome itself.

5

previous work has tended to focus on only a single aspect of tRNA function, such as

6

increased amber suppression or improved EF-Tu binding2,26, and did not take into account

7

changes in translational fidelity that could arise from mischarging. Selection experiments

8

that seek to optimize tRNA function must combine mutations from multiple regions for

9

maximal impact, and based on this work, we present a model MjY-tRNA library (Figure

10

S10) that can be used for selections aimed at optimizing the incorporation of non-canonical

11

amino acids.

However,

12

Most recent work for optimization of the Mj tyrosine suppressor pair has focused

13

upon evolution of the MjYRS7,11. We have shown that further gains for an expanded genetic

14

code are possible through the directed evolution of the heterologous tRNA. Previous

15

attempts to improve tRNA function employed traditional positive selection schemes (e.g.

16

antibiotic resistance selection) in conjunction with a strong negative selection (e.g.

17

barnase2,11). In this work, we present an alternative approach by implementing a negative

18

selection based on expression of mutant E. coli phenylalanyl-tRNA synthetase (PheS-

19

A294G) with relaxed substrate specificity12 in conjunction with proven CPR-based positive

20

selection10. Selection by mutant E. coli PheS production in the presence of a variable

21

concentration of p-Cl-Phe affords the benefits of a tunable negative selection, which allows

22

for gradually increasing stringency for orthogonal behavior by tRNA variants. Additionally,

23

CPR as a positive selection technique confers the benefit of exponential amplification found

24

in PCR, which ultimately leads to significantly higher enrichment per selection round. The

25

techniques employed herein for both tRNA selection and library design should be readily


Page 15 of 36

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1

adaptable to any target non-canonical amino acid, or to other heterologous synthetase and

2

tRNA pairs.

3

The generation of improved suppressor tRNA variants may have been a

4

consequence of the use of CPR, which occurs partially in vivo and partially in vitro, as a

5

selection method. During CPR, cells would have had to survive and propagate while

6

expressing variant tRNAs, but this initial selection for fitness should not have impacted the

7

recovery of functional tRNA variants during positive selection, because cells are lysed

8

following emulsification. This in turn led to a very stringent positive selection with the

9

enrichment of functional variants estimated to be roughly 103-fold per round of CPR

10

(Figure S1). In addition, we were able to introduce a new negative selection system, which

11

further refined the orthogonality of and likely the fitness costs accorded to suppressor

12

tRNAs. Variant S7 and its descendants can therefore be viewed as tRNAs that have been

13

more fully optimized for function, including for orthogonality and fitness, than tRNAoptCUA

14

and Nap1.

15

That said, fitness costs were only partially due to tRNA-mediated amino acid

16

incorporation into proteins. Even when incorporation was not programmed, due to a lack

17

of paired charging machinery (Figures 4A and S9) or to a lack of genomic amber codons

18

(the “Amberless” E. coli C321.ΔA; Figures 4B and 6), the selected tRNAs still proved to have

19

less of an impact on cell growth than previously selected variants. These results point to

20

other potential fitness costs in engineering translation, such as background mischarging or

21

competitive inhibition of other tRNA synthetases. Gratifyingly, CPR, especially when used in

22

conjunction with a negative selection, appears to be able to optimize for function and fitness

23

simultaneously. Overall, these selection experiments re-emphasize that multiple positions

24

in the compact tRNA molecule work together to balance efficiency, orthogonality, and



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Page 16 of 36

1

fitness, and that stringent directed evolution experiments can nonetheless lead to a

2

balancing of all of these different properties.

3 4

Materials and Methods

5

Cloning and amplification of plasmids was carried out in DH10B E. coli (Invitrogen).

6

Positive selection via CPR was carried out in BL21(DE3) E. coli strain.

7

“Amberless” E. coli C321.ΔA 8 was used for growth rate assays. All E. coli culture for both

8

cloning, selection, and assay occurred in 2xYT broth (Fisher Scientific).

9

concentrations were as follows: ampicillin (100 μg/mL), chloramphenicol (34 μg/mL), and

10

kanamycin (50 μg/mL). Plasmid constructs were sequence verified. All oligonucleotides

11

used in PCR amplication, sequencing, and library construction were synthesized by

12

Integrated DNA Technology (San Diego) and their sequences are listed in Table S3.

A strain of

Antibiotic

13 14

tRNA and tRNA-synthetase expression plasmids

15

We employed two previously designed plasmids for either sole expression of a tRNA gene

16

(pBRIVTC3B) or dual expression of tRNA and aminoacyl-tRNA synthetase genes (pRST) 9.

17

For plasmid pRST, the synthetase cassette was sequenced with forward and reverse oligos

18

ACM001 and ACM002. For both pBRIVTC3B and pRST plasmids, the tRNA cassette was

19

sequenced with forward and reverse oligos ACM003 and ACM004.

20 21

Assembly of tRNA libraries and variants

22

Libraries for tRNA selections were assembled by PCR amplification of a DNA Ultramer Oligo

23

(IDT). Combinatorial variants and other desired tRNA genes were similarly assembled from

24

amplification of an Ultramer Oligo (IDT). All Ultramer library and variant amplification was

25

done with tRNA-Lib_F and tRNA-Lib-R oligos.


Page 17 of 36

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1 2

Reporter and positive selection plasmid construction

3

All variants of Taq polymerase and superfast GFP (“sFastGFP”) 29 are flanked by 5’ NdeI and

4

3’ XhoI restriction sites (NEB). Taq polymerase and sFastGFP variants were cloned into

5

pACYCSolo 9, a T7-driven expression vector derived from pACYCDuet (Novagene). For

6

plasmid pACYCSolo, expression cassettes were sequenced with forward and reverse oligos

7

ACM031 and ACM032.

8 9

Codon optimized variants of Taq polymerase were assembled and amplified from multiple

10

gBlock gene fragments (IDT).

11

assembled containing four, six, eight, and ten amber codons. Amber codons replaced

12

surface exposed tyrosine residues as determined by the crystal structure of Taq polymerase

13

(1TAQ, pdb). DNA sequence information for Taq polymerase can be found in Table S4.

14

Variant Taq-4xAmb has four amber codons replacing surface exposed tyrosine residues

15

corresponding to Y24, Y45, Y116, and Y146. Derived from Taq-4xAmb, variant Taq-6xAmb

16

has six amber codons replacing surface exposed tyrosine residues corresponding to Y24,

17

Y45, Y116, Y146, Y161, and Y182. Derived from Taq-6xAmb, variant Taq-8xAmb has eight

18

amber codons replacing surface exposed tyrosine residues corresponding to Y24, Y45,

19

Y116, Y134, Y146, Y161, Y172, and Y182. Derived from Taq-8xAmb, variant Taq-10xAmb

20

has ten amber codons replacing surface exposed tyrosine residues corresponding to Y24,

21

Y45, Y116, Y146, Y161, Y182, Y339, and Y378.

Along with wild type Taq polymerase, variants were

22 23

Codon optimized variants of sFastGFP were amplified from single gBlock gene fragments

24

(IDT). Sequence information for sFastGFP can be found in Table S4. Along with wild type

25

sFastGFP, two variants were cloned containing one and three amber codons at surface-



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 36

1

exposed tyrosines as determined by the crystal structure of sfGFP (2B3P, pdb). Variant

2

sFastGFP-1xAmb replaced surface-exposed Y39 with an amber codon.

3

sFastGFP-3xAmb, three surface-exposed tyrosine residues – Y39, Y151, Y182 – were

4

replaced with amber codons.

For variant

5 6

Construction of pSELt vector

7

The kanamycin resistance (kanR) gene derived from pET28a plasmid. The leuQ promoter

8

driven tRNA expression cassette derived from pRST.B plasmid.

9

replication derived from pACYCSolo plasmid. The mutated constitutive trpL promoter as

The 15A origin of

10

well as E. coli pheS gene bearing a A294G mutation were amplified from pcat-pheS

11

whereas the intervening ACP gene derived from pACP (NEB). The linker sequence between

12

ACP and pheS-A294G containing two amber stop codons resulted from overlap extension of

13

primer overhangs. The sequence for pSELt can be found in Table S5 and its plasmid map is

14

in Figure S11.

30

15 16

Tyrosine suppressor tRNA selection by Compartmentalized Partnered Replication

17

(CPR)

18

The CPR protocol used for this work represents an optimized version of the original CPR

19

protocol 10.

20 21

Tyrosine tRNA libraries were cloned into pRST.B_66x.AA vector and transformed into

22

BL21(DE3) E. coli harboring pACYCSolo_Taq plasmid containing a predetermined number

23

of amber codons in the Taq polymerase gene. Each round of selection had transformation

24

efficiencies >107 to ensure complete coverage of the theoretical diversity from an initial

25

library. Following one-hour transformation recovery in SOC media, transformed cells were


Page 19 of 36

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1

diluted 1:10 in 2xYT media containing 2% glucose as well as ampicillin and

2

chloramphenicol. This transformation culture was shaken overnight at 37°C for twelve

3

hours. After twelve hours, a new culture was prepared from 4 mL of fresh 2xYT with

4

appropriate antibiotics into which 200 μL of overnight transformation culture was added.

5

This inoculated culture was shaken for one hour at 37°C after which IPTG was added for a

6

final concentration of 0.4 mM. Induced selection culture was shaken at 37°C for three

7

hours. After the three-hour expression, cells (300 μL) were spun down (15 min, 1500g,

8

4°C) and gently washed twice with chilled 1x Taq buffer (50 mM KCl, 10 mM Tris-HCl pH 8,

9

1.5 mM MgCl2). Washed cells were resuspended in 300 μL CPR buffer (50 mM KCl, 10 mM

10

Tris-HCl pH 8, 1.5 mM MgCl2, 200 μM dNTP, 0.3 nM each CPR primer (ACM 221 and

11

ACM222), 100 μM TMAC, and 0.05% RNaseI).

12 13

Resuspended cells were added to a pre-chilled 2 mL microtube containing: 876 μL Tegosoft

14

(Evonik), 240 μL mineral oil (Sigma), 84 μL AbilEW09 (Evonik), and the rubber tip from a 1

15

mL syringe. This mixture was allowed to rotate for five minutes in a cold room (4°C). The

16

cell-surfactant-oil mixture was emulsified using a TissueLyser LT (Qiagen), which was

17

operated at 42 Hz for four minutes in a cold room (4°C). Next, emulsified cells were thermal

18

cycled [95°C:3 min, 20 cycles of (95°C:30 s, 59°C:30 s, 72°C:90s/kb), 72°C:5 min, 4°C:∞] in

19

100 μL aliquots to amplify and recover functional tRNA variants. After thermal cycling,

20

emulsions were pooled on ice, spun down (10 min, 12,000g, 4°C), and the top layer of

21

excess oil was removed. The remaining emulsion was broken by applying an equal volume

22

of chloroform/isoamyl alcohol/phenol (24:1:25) (Sigma) and vortexing.

23

emulsion was then applied to a PhaseLock tube (5 Prime) and spun (2 min, 16,000g). The

24

top layer or aqueous fraction from this first extraction was pipetted into a fresh microtube

25

to which an equal volume of chloroform (Sigma) was added. This second extraction served


The broken


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

to remove residual organics and, after vortexing, it was placed in a new PhaseLock tube and

2

spun (2 min, 16,000g). The top layer or aqueous fraction from this second extraction was

3

collected and column purified (Zymo Research Corp). The elution from column purification

4

served as template for a secondary or recovery PCR using AccuPrime Pfx (Invitrogen) and

5

primers specific to the overhang regions of CPR primers used during emulsion PCR (see

6

Table S3). Resulting secondary or recovery PCR was run on a 0.8% agarose gel. The band

7

corresponding to the desired emulsion PCR amplicon was excised from the agarose gel and

8

extracted (Promega). This purified secondary amplicon was then used as template for a

9

third and final PCR, which generated sufficient template for cloning by restriction digest by

10

BsrGI and KpnI (NEB) for a subsequent selection round or for sequencing purposes.

11 12

Selection of 3-halo-tyrosine tRNA libraries closely resembled the tyrosine suppressor tRNA

13

selection scheme described above.

14

transformation, a new culture was prepared from 4 mL of fresh 2xYT containing 2 mM of

15

the appropriate 3-halo-tyrosine (3-Cl-Y, 3-Br-Y, 3-I-Y) with appropriate antibiotics into

16

which 200 μL of overnight transformation culture was added. This inoculated culture was

17

shaken for one hour at 37°C after which IPTG was added for a final concentration of 0.4 mM.

18

Following induction with IPTG and expression, cells were harvested and treated by the

19

same CPR protocol employed in tyrosine suppressor tRNA selection.

After the twelve-hour growth following library

20 21

Adapted pheS negative selection

22

For this adapted pheS negative selection, selection occurred in liquid media rather than on

23

solid agar plates. A tRNA library pool was cloned into pSELt_6x.A vector and transformed

24

into DH10B E. coli with transformation efficiencies of >107 to ensure complete coverage of

25

the theoretical diversity from an initial library.

Following one-hour transformation


Page 20 of 36

Page 21 of 36

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1

recovery in SOC media, transformed cells were diluted 1:10 in 2xYT media containing

2

kanamycin and shaken overnight at 37°C for no more than twelve hours. After twelve

3

hours, a selection culture was prepared from 4 mL of fresh 2xYT containing 10 mM p-Cl-Phe

4

(Sigma) and kanamycin into which 200 μL of overnight transformation culture was added.

5

This inoculated selection culture was shaken for four hours at 37°C. Next, 0.5 mL of this

6

first selection culture was passaged into a second culture tube of 4 mL 2xYT containing 10

7

mM p-Cl-Phe (Sigma) and kanamycin.

8

selection culture was spun down and miniprepped (Qiagen) to recover plasmid.

9

Miniprepped plasmid was used as template for PCR amplification using primers specific to

After another four-hour selection, this second

10

the tRNA cassette from pSELt.

11

restriction digest cloning by BsrGI and KpnI (NEB) for further selection.

PCR amplification product was used as template for

12 13

Design and selection of first-generation tRNA libraries: Ac-Lib and T-Lib

14

Initial libraries, Ac-Lib and T-Lib, were subjected to three rounds of CPR positive selection,

15

one round of pheS negative selection, and a final round of CPR positive selection. During the

16

first two rounds of CPR, library pools were expressed by lpp promoter and tasked with

17

suppressing Taq polymerase containing four amber codons. For the third round of CPR,

18

library pools were expressed by E. coli leuQ tRNA promoter and tasked with suppressing

19

Taq polymerase containing six amber codons. Library pools were then subjected to the

20

adapted pheS negative selection. A final round of CPR was conducted using the selection

21

conditions from round three of CPR. The final library pools were cloned and sequenced.

22 23

Selection of second-generation tRNA library: AcT5S -Lib



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

The AcT5S library was subjected to a similar, but more rigorous selection scheme than that

2

used with Ac-Lib and T-Lib. All selections for AcT5S were conducted with library pools

3

were expressed by E. coli leuQ tRNA promoter.

4 5

The first and second rounds of CPR tasked libraries with suppressing Taq polymerase

6

containing six and eight amber codons, respectively. The AcT5S library pool was then

7

subjected to one round of the adapted pheS negative selection. Two more rounds of CPR

8

selection were conducted with AcT5S library pool tasked with suppressing Taq polymerase

9

containing ten amber codons. The final library pool was cloned and sequenced.

10 11

Design and selection of third-generation tRNA libraries: S7-TA

12

Due to its smaller library size, the S7-TA library was only subjected to two rounds of CPR

13

positive selection. Each round tasked library pools with suppressing Taq polymerase

14

containing ten amber codons. Furthermore, three separate selections of the S7-TA library

15

were run in parallel with the three 3-halo-tyrosines: 3-chloro-tyrosine (3-Cl-Y), 3-bromo-

16

tyrosine (3-Br-Y), and 3-iodo-tyrosine (3-I-Y).

17 18

During the first round of CPR, selection of S7-TA followed the above-described protocol for

19

3-halo-tyrosine suppressor tRNA selection. However, during the second round of CPR, the

20

post-induction expression time was reduced from three hours to one hour in order to

21

increase selection stringency.

22

suppression activity screens. For each 3-halo-Y library pool, the activities of single colony

23

picks were assayed in comparison to tRNAoptCUA. The top eight picks from each 3-halo-Y

24

selection were sequenced and rescreened in triplicate to confirm tRNA performance. The

25

top two tRNA variants from each 3-halo-Y selection were used in further analyses.

The final library pools were cloned and subjected to


Page 22 of 36

Page 23 of 36

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1 2

Orthogonality assay protocol

3

Using chemically competent BL21(DE3) E. coli, transformations were conducted with the

4

reporter plasmid pACYCSolo_sFastGFP-1xAmb and pBRIVTC3B plasmid expressing the

5

tRNA variant to be assayed. Control samples resulted from the same double plasmid

6

transformation with the reporter plasmid pACYCSolo_sFastGFP-1xAmb and pBR322 lacking

7

a tRNA expression cassette. All transformations were plated on 2xYT agar petri dishes

8

containing ampicillin and chloramphenicol.

9 10

Using a 96-well 2 mL plate (Fisher), three distinct colonies from each sample petri dish

11

were picked into 1 mL of 2xYT media containing 2% glucose as well as ampicillin and

12

chloramphenicol. The 96-well plate was sealed with AirPore Tape (Qiagen) and shaken at

13

37°C for twelve hours. After twelve hours, 50 μL of the overnight culture was used to

14

inoculate 1 mL of fresh 2xYT media containing ampicillin and chloramphenicol in a new 96-

15

well 2 mL plate. The new plate was sealed with AirPore Tape and shaken at 37°C for one

16

hour. After one hour, assay cultures were induced by adding IPTG to each 1 mL culture for a

17

final concentration of 1 mM IPTG. Following induction with IPTG, the plate was seal with

18

AirPore Tape and shaken at 37°C for three hours.

19 20

After IPTG induction and expression, the assay plate was spun down (20 min, 3000g, 4°C)

21

and washed twice with 1xPBS. Next, 200 μL from each resuspended sample were pipetted

22

into a Costar Assay Plate, 96 Well Black with Clear Flat Bottom (Corning). Three wells with

23

200 μL 1xPBS served as a buffer or blank control. Measurements were taken with the

24

Infinite® 200 PRO plate reader (Tecan). Costar Assay Plate was gently shaken (3.5 mm



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Page 24 of 36

1

orbital amplitude) for ten seconds before measuring cell density with absorbance at 600 nm

2

(OD600) and in cell sFastGFP fluorescence intensity (Exc/Em: 485/528).

3 4

Tyrosine suppression efficiency assay protocol

5


6

reporter plasmid pACYCSolo_sFastGFP-3xAmb as well as pRST plasmid expressing both

7

wild type Mj tyrosyl-tRNA synthetase and the tRNA variant to be assayed. Negative control

8

samples resulted from the same double plasmid transformation with the reporter plasmid

9

pACYCSolo_sFastGFP-3xAmb and pBR322. Positive control samples resulted from double

10

plasmid transformation with reporter plasmid pACYCSolo_sFastGFP and pBR322.

11

transformations were plated on 2xYT agar petri dishes containing ampicillin and

12

chloramphenicol.

All

13 14

Following transformation of the above-mentioned plasmids with proper controls, the

15

tyrosine suppression efficiency assay was conducted with the same protocol employed in

16

the orthogonality assay.

17 18

Non-canonical incorporating suppression efficiency assay protocol

19


20

reporter plasmid pACYCSolo_sFastGFP-3xAmb as well as pRST plasmid expressing both 3-

21

iodo-tyrosine incorporating Mj synthetase and the tRNA variant to be assayed. Negative

22

control samples resulted from the same double plasmid transformation with the reporter

23

plasmid pACYCSolo_sFastGFP-3xAmb and pBR322.

24

without ambers were excluded from non-canonical incorporating suppression efficiency

25

assays. Instead, the relative activities of tRNA variants were compared to one another. All

Positive controls using sFastGFP


Page 25 of 36

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1

transformations were plated on 2xYT agar petri dishes containing ampicillin and

2

chloramphenicol.

3 4

Following transformation of the above-mentioned plasmids with proper controls, the non-

5

canonical incorporating suppression efficiency assay was conducted using the same

6

protocol employed in the orthogonality assay.

7 8

Fitness/Growth Assay protocol

9

DH10B and E. coli C321.ΔA cells containing tRNA expression plasmids (pBRIVTC3B

10

expressing variant tRNAs) or a pBR322 derivative lacking a tRNA expression cassette were

11

cultured overnight from single colonies. Cultures were diluted 1:100 and 10 μL used to

12

inoculate 200 μL fresh media in a 96-well Black with Clear Flat Bottom plate (Costar).

13

Following inoculation, cultures were sealed with Breathe-Easy sealing membrane (Sigma

14

Aldrich) and incubated at 37 °C with agitation (5 mm linear amplitude) in a preheated plate

15

reader (Infinite 200 PRO, Tecan). OD600 measurements were taken every 900 seconds for a

16

period of 16.5 hours.

17 18

Supporting Information

19

Pertinent tRNA sequences (Table S1), agarose gel representing the results of mock CPR

20

positive selections (Figure S1), graphical representation of tRNA variants from first-

21

generation library selections (Figure S2), map of extra-library mutations from tRNA

22

variants from first-generation library selections (Figure S3), graphical table detailing the

23

design of the ten combinatorial variants generated from top tRNA variants of both first-

24

generation library selections (Figure S4), in vivo suppression efficiency and orthogonality

25

assays for combinatorial variants and parental tRNAs (Figure S5), graphical representation



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 26 of 36

1

of second-generation library based on AcT.05 (Figure S6), calculated parameters from

2

growth curves fitted with a modified logistic growth model for DH10B and “Amberless”

3

C321.∆A E. coli (Table S2), in vivo suppression efficiency assay for most active variants from

4

second-generation library (Figure S7), graphical representation of the mutations for

5

second-generation library variants assayed in Figure S7 (Figure S8), growth assay in DH10B

6

E. coli of most pertinent tRNA variants (Figure S9), proposed library for selecting new tRNA

7

variants for optimizing EF-Tu binding for a given unnatural amino acid (Figure S10), table

8

of pertinent oligos and their sequences (Table S3), table of sequences for codon optimized

9

genes used in selections (Table S4), nucleotide sequence for pSELt plasmid with tRNAoptCUA

10

(Table S5), plasmid map for pSELt with tRNAoptCUA (Figure S11)

11 12

Abbreviations

13

Orthogonal translation system (OTS), tRNA synthetase (RS), aminoacyl-tRNA synthetase

14

(aaRS), compartmentalized partnered replication (CPR), non-canonical amino acid (ncAA)

15 16

Author Information

17

Corresponding

18

[email protected]. AM designed and executed all selections and screening assays.

19

AM and AE wrote the paper.

Author:

Tel:

(512)

471-6445.

Fax:

(512)

471-7014.

E-mail:

20 21

Acknowledgement

22

The authors declare no competing interests. We thank Dr. Ross Thyer and Dr. Randall

23

Hughes for contributing materials in the form of plasmid constructs. This work by the

24

Welch Foundation (Project # F-1654) and by the Air Force and Department of Defense


Page 27 of 36

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1

through the National Security Science and Engineering Faculty Fellowship (Project #

2

FA9550-10-1-0169) and the Air Force Research Lab AFOSR (Project # FA9550-14-1-0089).

3



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1

References

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Abstract Graphic 114x78mm (300 x 300 DPI)


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General schematic representing compartmentalized partnered replication (CPR) as a two-plasmid system. In this scheme, the function of the partnered gene (green and red boxes) is coupled to the expression of Taq polymerase forming an inducible genetic circuit. The partnered gene is represented as a diverse library pool of varying activity (red indicates low or nonfunctional, green color indicates higher activity). The library pool for a partner gene is cloned into its corresponding expression plasmid and transformed into E. coli bearing the Taq polymerase expression plasmid. Overnight culture is used to inoculate fresh media containing all components necessary for the selection. Then the genetic circuit is induced and allowed to express for some time. Cells with functional or higher activity partnered gene variants will produce more Taq polymerase whereas cells with nonfunctional partnered genes will not produce any Taq polymerase. Following expression, cells are washed, resuspended in a PCR mix containing primers for amplification of the partnered gene, and emulsified in an oil/surfactant mixture. The resulting emulsions are thermocycled. A bacterial cell bearing a functional or higher activity partnered gene will release expressed Taq polymerase into its emulsion bubble allowing amplification of that partnered gene variant. No amplification will occur in the emulsion bubble of a bacterial cell bearing a nonfunctional partnered gene as it did not express Taq polymerase. In this manner, functional and higher activity variants of the partnered gene are greatly enriched during each round of CPR selection. 86x61mm (300 x 300 DPI)



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Characterization of selected tyrosine suppressor tRNAs. (A) Structure and sequence for tRNAoptCUA flanked by segments representing the randomized sequences from first-generation libraries: Ac-Lib (lower-left) & TLib (upper-right). (B) Variant AcT.05 tRNA. This tRNA is labeled to demonstrate mutations that derived from Ac-Lib (blue) and T-Lib (yellow) libraries, as well as non-library mutations (orange) that arose. Dark shading indicates nucleotide changes while light shading indicates randomized nucleotides that reverted to the original tRNAoptCUA sequence. (C) Variant S7 tRNA. Coloring indicates mutations that derived from Ac-Lib (blue), T-Lib (yellow), and AcT5S (green) libraries. Dark shading indicates nucleotide changes while light shading indicates randomized nucleotides that reverted to the tRNAoptCUA sequence. (D) In vivo suppression by evolved tyrosine tRNAs and their progenitor, tRNAoptCUA, with the fluorescent reporter sFastGFP containing three amber codons. Values represent the averages of triplicate RFU/OD600 readings with error bars representing standard errors. All measurements and errors have been normalized to the wild-type sFastGFP that contained no amber codons. (E) Orthogonality assay for evolved tyrosine tRNAs and their progenitor, tRNAoptCUA, using the fluorescent reporter sFastGFP containing one amber codon. Values represent the


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averages of triplicate RFU/OD600 readings with error bars representing standard errors. All values have been normalized to the pBR322 control. 155x228mm (300 x 300 DPI)



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Characterization of halo-tyrosine suppressor tRNAs. (A) S7-TA library based on variant S7. The structure is labeled with tRNAoptCUA reversions (beige), mutations maintained from previous selections (grey), and randomized positions (red). (B) Proposed S7-TA2 library based on the results from selections with the S7TA library for 3-halo-tyrosine incorporation. The structure is labeled with tRNAoptCUA reversions (beige), mutations maintained from previous selections (grey), and randomized positions (red). (C) Acceptor stem and T-arm sequences for the S7-TA library and the five 3-halo-tyrosine incorporating tRNAs variants. Each tRNA name has been color-coded to indicate the selection from which it was isolated: 3-I-Y (purple), 3-Br-Y (red), and 3-Cl-Y (green). (D) Suppression assay for 3-halo-tyrosine incorporation with the fluorescent reporter sFastGFP containing three amber codons. Each tRNA variant was assayed in the presence of 3iodo-tyrosine (purple), 3-bromo-tyrosine (red), and 3-chloro-tyrosine (green). Values represent the average of three samples measuring RFU/OD600 with error bars representing standard errors. All values were normalized to those obtained for tRNAoptCUA. (E) Orthogonality assay for the five 3-halo-tyrosine incorporating tRNAs and comparable tyrosine suppressor tRNAs (S7, Nap1 and tRNAoptCUA) using the fluorescent reporter sFastGFP containing one amber codon. Values represent the averages of triplicate RFU/OD600 readings with error bars representing standard errors. All values were normalized to the pBR322 control. 152x135mm (300 x 300 DPI)


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Growth assays for tRNA variants expressed without a paired aaRS under the control of the leuQ promoter. Assays were conducted in DH10B (A) and “Amberless” C321.∆A E. coli (B) grown in 2xYT media. Black diamonds indicate growth in the presence of the control plasmid pBR322 containing no tRNA expression cassette. Blue circles indicate the expression of Nap1. Red squares indicate the expression of tRNAoptCUA. Green triangles indicate the expression of tRNA variant S7. Error bars show the standard deviations for triplicate assays. 96x137mm (300 x 300 DPI)



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Growth assays in DH10B with wild-type and halotyrosine-incorporating amino acyl tRNA synthetases. Each growth assay is arranged in a grid format whereby the media conditions vary for each row and the columns indicate which Mj synthetase variant was expressed. The top row represents growth in 2xYT media. The middle row represents growth in 2xYT media with 2% glucose. The bottom row represents growth in 2xYT media with 0.1 mM IPTG. Each synthetase was expressed with the tacI promoter and the tRNA variants were expressed under the control of the leuQ promoter. The left-hand column are experiments with the wild-type MjYRS and the right-hand column are experiments with a Mj synthetase variant capable of incorporating 3-halo-tyrosines21. Blue circles indicate growth with expression of Nap1. Red squares indicate growth with expression of tRNAoptCUA. Green triangles indicate growth with expression of tRNA variant S7. 150x169mm (300 x 300 DPI)


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Growth assays in C321.∆A with wild-type and halotyrosine-incorporating amino acyl tRNA synthetases. The layout is as in Figure 5. 151x168mm (300 x 300 DPI)


Evolving Orthogonal Suppressor tRNAs To Incorporate Modified

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