Supplementary Figure 1

Selenocysteine biosynthesis is toxic and incorporation is poorly maintained.

Dilution plating of RTΔA cells transformed with pRSF-UX-SelA (wild-type or truncated) and p15A-NMC-A (wild-type or C69U-C238U) demonstrates two related phenomena; toxicity of selenocysteine biosynthesis and loss of selenocysteine incorporation. In addition to expressing the NMC-A β-lactamase, p15A-NMC-A confers tetracycline resistance. (a-b) The presence of functional SelA (panel a) drastically decreases cell growth and viability following transformation compared to cells which lack SelA (panel b). Carbenicillin

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resistance is directly correlated with tetracycline resistance. (c-d) Cells transformed with NMC-A C69U-C238U and functional SelA can grow in the presence of carbenicillin (panel c), while those with a truncated version of SelA do not (panel d). However, carbenicillin resistant cells are 10-fold less abundant than tetracycline resistant cells (panel c) indicating not all transformants are capable of

incorporating selenocysteine (at levels sufficient to support β-lactamase activity). Plates are representative from two biologically independent series.

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Supplementary Figure 2

Growth curves in rich media (LB) of parental strains and descendant populations evolved under β-lactam stress.

The labels indicate the carbenicillin concentration and the curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. (a) Δ parental strain and evolved populations β_Δ1-3. (b) CC parental strain and evolved populations β_CC1-3. (c) UC parental strain and evolved populations β_UC1-3. (d) UU parental strain and evolved

populations β_UU1-3. Evolved populations have increased growth rate at given carbenicillin concentrations, reach higher culture density and show greater resistance to carbenicillin compared to their respective parental strains.

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Supplementary Figure 3

Growth curves in selenium free media (MOPS EZ) of parental strains and descendant populations evolved under β-lactam stress.

The labels indicate the media additives and the curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. Carbenicillin and Na2SeO3 were added at concentrations of 100 μg.mL and 1 μM respectively. (a) Δ parental strain and evolved populations β_Δ1-3. (b) CC parental strain and evolved populations β_CC1-3. (c) UC parental strain and evolved populations β_UC1-3. (d) UU parental strain and evolved populations β_UU1-3. The Δ and UU parental

strains had severely impaired growth in MOPS EZ media, particularly when grown in 96-well plates. Evolved populations have increased growth rate and reach higher culture density compared to parental strains. All β_UC and β_UU populations retained selenocysteine dependence, requiring selenite supplementation to grow in the presence of carbenicillin.

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Supplementary Figure 4

Growth curves in rich media (LB) at 37 °C of parental strains compared to descendant populations evolved under thermal stress.

The curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. (a) Δ parental strain compared to evolved populations T_Δ1-3. (b) CC parental strain compared to evolved populations T_CC1-3. (c) UC parental strain compared to evolved populations T_UC1-3. (d) UU parental strain compared to evolved populations

T_UU1-3. Evolved populations generally had poorer growth rates and reached lower culture density compared to parental strains.

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Supplementary Figure 5

Growth curves in selenium free media (MOPS EZ) of parental strains and descendant populations evolved under thermal stress.

The labels indicate the media additives and the curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. Carbenicillin and Na2SeO3 were added at concentrations of 100 μg.mL and 1 μM respectively. (a) Δ parental strain and evolved populations T_Δ1-3. (b) CC parental strain and evolved populations T_CC1-3. (c) UC parental strain and evolved populations T_UC1-3. (d) UU parental strain and evolved populations T_UU1-3. The Δ and UU parental

strains had severely impaired growth in MOPS EZ media, particularly when grown in 96-well plates. Evolved populations have increased growth rate and reach higher culture density compared to parental strains. All T_UC and T_UU populations retained selenocysteine dependence, requiring selenite supplementation to grow in the presence of carbenicillin.

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Supplementary Figure 6

Mass spectrometry of GFP X177 expressed in β_Δ1 and T_Δ1 cells reveals that UAG codons are read as glutamine by near cognate suppression.

(a) Mass spectrum of intact GFP X177 expressed in β_Δ1 cells. (b) Deconvoluted mass spectrum of GFP X177 expressed in β_Δ1 cells reveals two species resulting from partial loss of the N-terminal methionine residue. (c) Experimentally determined monoisotopic

masses (27928.91 Da and 27797.87 Da) match the theoretical masses of (27928.85 Da and 27797.81 Da) of GFP containing a glutamine residue at position 177. (d) Mass spectrum of intact GFP X177 expressed in T_Δ1 cells. (e) Deconvoluted mass spectrum of GFP X177 expressed in T_Δ1 cells. (f) Experimentally determined monoisotopic masses (27928.91 Da and 27797.88 Da) match the

theoretical masses of (27928.85 Da and 27797.81 Da) of GFP containing a glutamine residue at position 177. All spectra are representative from a minimum of two technical replicates.

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Supplementary Figure 7

Growth curves of RTΔA cells containing point mutants in oxidative stress and selenite resistance.

Growth curves of RTΔA cells containing point mutants in oxidative stress and selenite resistance genes in rich and defined media ± 1 μM Na2SeO3. The curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. (a) Growth curves of RTΔA containing a constitutively active variant of OxyR (A233T) observed during evolution. The A233T mutation does not provide any clear benefits to cell fitness compared to a wild-type control. (b-d) Growth curves of RTΔA

containing T69I, T73A or H153Y mutations respectively in CysK, which are expected to strongly inhibit cysteine biosynthesis, compared to a wild-type control. Mutations are not observed to have a significant impact on cell growth.

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Supplementary Figure 8

Mutant cysK allele retention and plasmid copy number determination.

Analysis of mutant cysK allele frequency by qPCR in cell populations passaged for 125 generations in the presence of 10 μM Na2SeO3. At generation 0 (purple bars), cell populations contained 50% wild-type cysK and 50% mutant cysK. Bars represent the mean ± S.E.M. (a) Detection of wild-type cysK (T69) in WT:T69I populations ± Na2SeO3. Wild-type cysK is detected at a similar frequency in both the presence and absence of Na2SeO3. (b) In contrast, mutant T69I undetectable (Cq = ~ 35) after 125 generations in all populations. (c) Detection of wild-type cysK (T73) in WT:T73A populations does not change ± Na2SeO3. (d) The cysK T73A mutant is lost from the

populations when cultured in the absence of Na2SeO3 (light blue). In contrast, in the presence of Na2SeO3 (dark blue) the mutant allele is retained. (e) Detection of wild-type cysK (H153) in WT:H153Y populations is not affected by Na2SeO3 treatment. (f) Similar to T73A,

the H135Y mutant is undetectable after serial passage in media without selenite (light blue), but is partially retained when selenite is present (dark blue). Abundance of pCDF (g) and pRSF (h) plasmids in RTΔA cells with mutations in poly(A) polymerase I (pcnB) and DNA polymerase I (polA). All mutations significantly decreased plasmid abundance compared to wild-type cells. Bars represent the mean ± S.E.M.

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Supplementary Figure 9

Growth curves of C321.ΔA and prfB (release factor 2) single point mutants in RTΔA cells in rich and defined media.

The curves are plotted with a line showing the mean and shading representing ± S.E.M. where n = three independent biological replicates. (a) Growth of RTΔA ftsA G124E shows minor improvement in defined media. (b) Growth of RTΔA hemA P196L shows significant improvement in defined media. (c) No improvement was observed in either rich or defined media for RTΔA pta P673L. (d) Growth of RTΔA yeeJ S1467P shows significant improvement in defined media. (e) The T246A mutation in release factor two significantly improves growth of RTΔA cells in defined media. (f) No improvement was observed in either rich or defined media for RTΔA prfB N276D. (g) A T246A-N276D double mutant was generated to investigate potential synergy between T246A and N276D which co-occurred in one evolved population. Observed growth improvement is due to T246A mutation only. (h) Growth of RTΔA prfB

K282R shows minor improvement in defined media.

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Supplementary Figure 10

Mass spectrometry of GFP X177 expressed in β_CC1 and T_CC1 cells in the absence of Na2SeO3 confirms the incorporation of serine.

(a) Mass spectrum of intact GFP X177 expressed in β_CC1 cells. (b) Deconvoluted mass spectrum of GFP X177 expressed in β_CC1 cells reveals two species resulting from partial loss of the N-terminal methionine residue. (c) Experimentally determined monoisotopic

masses (27887.87 Da and 27756.84 Da) match the theoretical masses of (27887.83 Da and 27756.79 Da) of GFP containing a serine residue at position 177. (d) Mass spectrum of intact GFP X177 expressed in T_CC1 cells. (e) Deconvoluted mass spectrum of GFP X177 expressed in T_CC1 cells. (f) Experimentally determined monoisotopic masses (27887.85 Da and 27756.84 Da) match the

theoretical masses of (27887.83 Da and 27756.79 Da) of GFP containing a serine residue at position 177. All spectra are representative from a minimum of two technical replicates.

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Supplementary Figure 11

Fluorescence screen for retention of selenocysteine incorporation in evolved populations using seleno-smURFP.

Internal controls are located below the streaks are (from left to right) ΔsmURFP, smURFP-C52, seleno-smURFP (U52) and smURFP-S52. (a) β_Δ populations deficient in selenocysteine incorporation were non-fluorescent. Both the non-selenocysteine dependent population (β_CC) and the two selenocysteine dependent populations (β_UC and β_UU) had almost complete retention of the trait. (b)

T_Δ populations deficient in selenocysteine incorporation were non-fluorescent. The selenocysteine dependent T_UC and T_UU populations had almost complete retention of the trait, although no transformants could be obtained from T_UC3 and only a small number from T_UU3. In contrast, retention was highly variable across the three T_CC populations which were not selenocysteine dependent. Note, in this composite image the T_UU2 plate was imaged after an additional 24 hours of incubation at 37 °C due to very slow growth of the streaked cells using an exposure time of 0.5 seconds. The internal controls are consistent with the other plates and clear differences are observed between the streaked cells and the non-fluorescent smURFP-S52 control.

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Supplementary Figure 12

Mass spectrometry of two recombinant selenoproteins expressed in β_UU3 cells confirms highly efficient selenocysteine incorporation and correct formation of a diselenide bond.

(a) Mass spectrum of intact EcDHFR U39-U85. (b) Deconvoluted mass spectrum of EcDHFR U39-U85. (c) Experimentally determined monoisotopic mass (19054.13 Da) matches the theoretical mass (19054.14 Da) of EcDHFR U39-U85 containing a diselenide bond. The experimental mass has been adjusted to account for deconvolution error arising from the unusual isotope distribution of selenium. (d) UVPD mass spectrum of the 17+ charge state. (e) UVPD fragment map shows a lack of detectable fragments between U39 and U85 which confirms diselenide bond formation. (f) Mass spectrum of intact GFP U135-U177. (g) Deconvoluted mass spectrum of GFP U135-U177. (h) Experimentally determined monoisotopic masses (28013.64 Da and 27882.61 Da) match the theoretical masses of

(28013.66 Da and 278861 Da) of GFP U135-U177 containing a diselenide bond ± the N-terminal methionine residue. The experimental masses have been adjusted to account for deconvolution error. (i) UVPD mass spectrum of the 28+ charge state. (j) UVPD fragment

map shows a lack of detectable fragments between U135 and U177 which confirms diselenide bond formation. The lack of fragments near Y66 corresponds to the mature GFP chromophore. All spectra are representative from a minimum of two technical replicates.

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Supplementary Figure 13

Mass spectrometry of two recombinant selenoproteins expressed in β_UU3 cells confirms diselenide bonds can replace essential structural disulfide bonds in therapeutically relevant protein families.

(a) Mass spectrum of intact seleno-anti-MS2 scFv. (b) Deconvoluted mass spectrum of seleno-anti-MS2 scFv. (c) Experimentally

determined monoisotopic mass (31855.08 Da) matches the theoretical mass (31855.06 Da) of seleno-anti-MS2 containing a two diselenide bonds. The experimental mass has been adjusted to account for deconvolution error arising from the unusual isotope distribution of selenium. (d) UVPD mass spectrum of the 24+ charge state. (e) UVPD fragment map shows a lack of detectable

fragments between U42 and U116 in the VH domain, and U179 and U249 in the VL domain, which confirms correct formation of both diselenide bonds. (f) Mass spectrum of intact seleno-hGH. (g) Deconvoluted mass spectrum of seleno-hGH. (h) Experimentally

determined monoisotopic mass (23400.27 Da) matches the theoretical mass (23499.27 Da) of seleno-hGH containing two diselenide bonds. The experimental mass has been adjusted to account for deconvolution error. (i) UVPD mass spectrum of the 16+ charge state. (j) UVPD fragment map shows a lack of detectable fragments between U54 and U166 and U183 and U190 which confirms correct

formation of both diselenide bonds. All spectra are representative from a minimum of two technical replicates.

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Supplementary Figure 14

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Mass spectra of Herceptin (Trastuzumab) scFvs.

(a) Mass spectrum of intact wild-type Herceptin-scFv expressed under reducing conditions. (b) Deconvoluted mass spectrum of wild-type Herceptin-scFv. (c) Experimentally determined monoisotopic mass (27172.05 Da) matches the theoretical mass (27172.08 Da) of wild-type Herceptin scFv containing reduced cysteine residues. (d) UVPD mass spectrum of the 29+ charge state of wild-type Herceptin. (e) UVPD fragment map shows extensive fragmentation across both the VL domain, and the VH domain, which confirms a lack of disulfide bonds. (f) Mass spectrum of intact wild-type Herceptin-scFv expressed under oxidizing conditions. (g) Deconvoluted mass spectrum of wild-type Herceptin scFv. (h) Experimentally determined monoisotopic mass (27170.12 Da) matches the theoretical mass (27170.07 Da) of wild-type Herceptin-scFv containing a single disulfide bond. (i) UVPD mass spectrum of the 23+ charge state of wild-type Herceptin-scFv. (j) UVPD fragment map shows extensive fragmentation across the VL domain, but a large fragment gap between C149 and C223 in the VH domain, which confirms the formation of one disulfide bond. (k) Mass spectrum of intact seleno-Herceptin-scFv expressed in β_UU3 cells. (l) Deconvoluted mass spectrum of seleno-Herceptin-scFv. (m) Experimentally determined

monoisotopic mass (27359.88 Da) matches the theoretical mass (27359.83 Da) of seleno-Herceptin-scFv containing two diselenide bonds. The experimental mass has been adjusted to account for deconvolution error arising from the unusual isotope distribution of selenium. (n) UVPD mass spectrum of the 20+ charge state of seleno-Herceptin-scFv. (o) The UVPD fragment map shows a lack of detectable fragments between U24 and U89 in the VL domain, and U149 and U223 in the VH domain, which confirms correct formation

of both diselenide bonds. All spectra are representative from a minimum of two technical replicates.

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Supplementary Figure 15

Mass spectra of anti-RCA scFvs.

(a) Mass spectrum of intact wild-type anti-RCA scFv. (b) Deconvoluted mass spectrum of wild-type anti-RCA scFv. (c) Experimentally

determined monoisotopic mass (27002.21 Da) matches the theoretical mass (27002.17 Da) of wild-type anti-RCA scFv containing a two disulfide bonds. (d) UVPD mass spectrum of the 20+ charge state. (e) The UVPD fragment map shows a lack of detectable fragments between C23 and C97 in the VH domain, and C159 and C224 in the VL domain, which confirms correct formation of both disulfide bonds. (f) Mass spectrum of intact seleno-anti-RCA. (g) Deconvoluted mass spectrum of seleno-anti-RCA. (h) Experimentally

determined monoisotopic mass (27193.96 Da) matches the theoretical mass (27193.94 Da) of seleno-anti-RCA containing two diselenide bonds. The experimental mass has been adjusted to account for deconvolution error arising from the unusual isotope distribution of selenium. (i) UVPD mass spectrum of the 20+ charge state. (j) The UVPD fragment map shows a lack of detectable fragments between U23 and U97 in the VH domain, and U159 and U224 in the VL domain, which confirms correct formation of both

diselenide bonds. All spectra are representative from a minimum of two technical replicates.

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