Materials and Reagents

Consumables

14 mL culture tubes (Fisher Scientific, Fisherbrand^TM, catalog number: 149566B)
Petri dishes (Fisher Scientific, Fisherbrand^TM, catalog number: FB0875713)
Multichannel reservoir (Fisher Scientific, Biotix^TM, catalog number: 12111089)
Deep well 96-well plates (Fisher Scientific, Fisherbrand^TM, catalog number: 12566611)
Black walled 96-well microplates (Fisher Scientific, Greiner-Bio, catalog number: 07-000-166)
Breathable microplate seal covers (Fisher Scientific, Andwin Scientific, catalog number: NC1660916)
1 mL cuvettes (Fisher Scientific, Fisherbrand^TM, catalog number: 14955128)
PCR strips (Fisher Scientific, BrandTech, catalog number: 14380941)
Serological pipettes (Fisher Scientific, Basix, catalog number: 14-955-235)
15 mL conical tubes (Fisher Scientific, Basix, catalog number: 14955237)
Inoculation loops/toothpicks (Loops: Fisherbrand^TM, catalog number: 22-363-602)

B. subtilis strains are available here: https://bgsc.org/search.php?Search=bDS
Associated annotated DNA sequences are available in the supplemental data to the original Nature communications publication: https://static-content.springer.com/esm/art%3A10.1038%2Fs41467-021-25691-4/MediaObjects/41467_2021_25691_MOESM6_ESM.zip

Non-standard Amino Acids

L-4,4-Biphenylalanine (BipA) (Peptech, catalog number: AL506)
L-4-Azidophenylalanine (pAzF) (abcr-GmbH, catalog number: AB308874)
Coumarin-nsAA (CouAA) (Sigma-Aldrich, catalog number: 792551)
L-4-Benzoylphenylalanine (BpA) (Peptech, catalog number: AL660)
L-4-Boc-lysine (boc-K) (Chem Impex Int’l, catalog number: 00363)
L-5-Hydroxytryptophan (5OHW) (Sigma-Aldrich, catalog number: 107751)
L-4-methyl-phenylalanine (4MeF) (Peptech, catalog number: AL096)
L-4-propargyloxy-phenylalanine (pPrF) (Combi-Blocks, catalog number: QW-3179)
L-4-amino-phenylalanine (4AmiF) (Peptech, catalog number: AL305)
L-4-aminomethyl-phenylalanine (4AmiMeF) (Peptech, catalog number: AL300)
L-benzenepentanoic acid, alpha-amino (BzpA) (Peptech, catalog number: AL514)
L-4-Nitro-phenylalanine (pNitroF) (Peptech, catalog number: AL061)
L-4-cyano-phenylalanine (pCNF) (Fisher Scientific, catalog number: AAH63572MD)
L-4-Fluoro-phenylalanine (pFF) (Peptech, catalog number: AL021)
L-4-Iodo-phenylalanine (pIF) (Peptech, catalog number: AL261)
L-4-Acetyl-phenylalanine (pAcF) (Peptech, catalog number: AL624)
L-4-methoxy-phenylalanine (4MeOF) (Alfa aesar, catalog number: H63096)
L-2-Naphthylalanine (NapA) (Peptech, catalog number: AL121)
L-bipyridyl-phenylalanine (biPyrA) (No longer commercially available)
L-4-tert-Butyl-tyrosine (tBut-Y) (Sigma-Aldrich, catalog number: 533130)

Additional Reagents

IPTG (Thermo Scientific^TM, catalog number: FERR0392)
Glucose (Thermo Scientific^TM, catalog number: AA1109036)
Glutamate (Acros Organics, catalog number: 156211000)
Ammonia sulfate (Sigma-Aldrich, catalog number: A4418)
Magnesium chloride, anhydrous (Sigma-Aldrich, catalog number: M8266)
Calcium chloride, dihydrate (Sigma-Aldrich, catalog number: 223506)
Manganese chloride (Sigma-Aldrich, catalog number: 416479)
Zinc chloride, anhydrous (Fisher Scientific, catalog number: AA1235722)
Thiamine-HCl (Sigma-Aldrich, catalog number: T1270)
Hydrochloric acid (10N) (RICCA Chemical Company, catalog number: 3770-32)
Iron (III) chloride, anhydrous (Fisher Scientific, catalog number: AAA1628122)
Potassium phosphate (monobasic) (Sigma-Aldrich, catalog number: P9791)
MOPS (free acid) (Fisher Scientific, TCI America, catalog number: M0707)
Chloramphenicol (Sigma-Aldrich, catalog number: C0378)
Kanamycin sulfate (Sigma-Aldrich, catalog number: K4000)
Phosphate buffered saline (tablet form) (Sigma-Aldrich, catalog number: P4417)
Sodium hydroxide (Acros Organics, catalog number: 134070010)

Media

LB Broth, Lennox (Fisher Scientific, Fisher BioReagents^TM, catalog number: BP9722500)
LB Agar, Lennox (Fisher Scientific, Fisher BioReagents^TM, catalog number: BP9745500)
S750 (1 L) (see Recipes)
ammoniaS750 (1 L) (see Recipes)
10× S750 salts (1 L) (see Recipes)
100× metals (500 mL) (see Recipes)

Equipment

Note: Equipment specifications are largely recommendations as this protocol has been validated for a series of different instruments.

-80 °C freezer
Incubator (Thermo Scientific, catalog number: 51-028-065HPM) or Shaking Incubator (Infors-HT Multitrons)
Microplate readers (protocol performed on both the Molecular Devices SpectraMax i3X or BioTek H1M Synergy)
Spectrophotometer (Thermo Scientific, catalog number: 840301000)
Plate shaker (Thermo Scientific, catalog number: 88882005)
Rotor drum (FisherBrand, catalog number: 14-251-251)
Pipettes (Fisher Scientific, Eppendorf Research Plus)
Pipettor (Fisher Scientific, Eppendorf EasyPet 3, catalog number: 12-654-105)
Vortex mixer (Scientific Industries, Inc. Vortex-Genie 2, catalog number: SI-0236)

Software

Excel
Software associated with plate reader used

Procedure

S750 protocol for tubes and 96-well plates
1. Remove glycerol stock that contains desired B. subtilis strain from -80 °C freezer. Streak cells from the glycerol stock onto an LB agar plate. Antibiotics may be useful for confirming strain identity but are not necessary as incorporation constructs are stable integrations. Incubate overnight at 37 °C. See Note 1 for essential controls that should be included in every experiment.
2. On the next day, pick desired colonies from the LB plate and transfer to 1 mL of S750 media in culture tubes. Mix vigorously by pipetting up and down to break up biofilms and promote suspension growth. These serve as the seed cultures for the experiment, without induction of heterologous gene expression. See Note 2 for optional media conditions.
3. Grow in a 37 °C shaking incubator or rotor drum for 4–6 h, to an OD between 0.2 and 0.7.
4. Prepare experimental media (which will serve to induce heterologous gene expression and nsAA incorporation) by adding the desired IPTG and nsAA to S750. IPTG final concentration is 1 mM, which is 1,000× of a 1 M stock solution. IPTG is typically added to all cultures including nsAA, except in the case of titration; for double titration see Figure 1. nsAA final concentration is usually 100 μM for most tyrosine-derived nsAAs. 1 mM should be used for boc-K, 5OHW, and CouAA. Similarly, other concentrations can be used for titration (see Figure 1 for an example). Different nsAAs will exhibit different titration dynamics due to the differences in binding to their cognate synthetases.
  1. For a 96-well plate format, a suggested higher throughput strategy is to prepare the master mix media unique to each nsAA in 15 mL tubes, then transfer to reservoir and distribute 300 μL with a 1 mL multichannel into a 2 mL deep well plate. In our experience, culture volumes above 300 μL do not aerate well in the deep 96-well plate format. See Figure 2 for the suggested media layout.
  2. For single culture tubes, use no more than 1 mL media per tube for aeration reasons.
  Figure 1. Double titration with nsAAs and transcriptional inducers. A UAG-mNeongreen construct under the control of a pHyperspank promoter was expressed with variable levels of nsAA and IPTG; a very sensitive induction was possible with low levels of IPTG and titration of nsAA. The signal was normalized to a positive control construct, where a TAC tyrosine codon replaced the UAG stop codon and 1 mM IPTG was provided. For more details, see Data analysis.
  
  Figure 2. Suggested plate layout for easy plate-filling. This plate layout is easy to set up with a multichannel pipette with minimal errors.
5. Measure OD of the seed cultures, then dilute them with additional S750 to start the experimental cultures at a theoretical density of 0.002 with desired cells.
  1. For a 96-well plate setup, dilute cultures with S750 in a PCR tube to OD 0.2, then use a multichannel to distribute 3 μL across the plate as desired, to achieve a final OD of 0.002 in 300 μL of experimental media.
  2. For single culture tubes, dilute media appropriately to final OD 0.002. For example, add 4 μL of OD 0.5 seed culture to 1 mL of S750 containing IPTG and nsAA.
6. Incubate tubes/plate overnight, shaking at 37 °C.
  1. For 96-well deep well plates, cover with a breathable cover (see Note 4b). Example data are in Figure 3a.
  2. For clear-bottomed shallow 96-well plates used in time courses, cover with a transparent breathable cover. Shaking should be set to a fast speed, though either orbital or double orbital can be used. Read OD₆₀₀ and fluorescence every 5–10 min. For the mNeongreen reporter, reading at 488/530 is acceptable, though many different filter settings will work for mNeongreen. Example data in Figure 3b.
7. For single culture tubes, grow in a 37 °C shaking incubator or rotor drum.
8. Read endpoint data after overnight growth. Extensively long growth periods will see some decrease of signal, but anything between 14 and 24 h is usually adequate.
  1. For fluorescence readout, dilute culture 1:1 with PBS, mix thoroughly, transfer 200 μL to clear-bottomed plates, and read OD and fluorescence. If the nsAA used is itself fluorescent, such as CouAA, spin down cells and wash them two to three times in PBS before resuspending in PBS for the plate reader experiment. If attempting microscopy, do washes in 1.5 mL tubes, carefully removing as much of the wash as possible in each case, and thoroughly resuspend pellets by pipetting. CouAA’s broad fluorescence spectrum grants very high background unless it is sufficiently washed out.
  2. For nanoLuciferase readout, add 100 μL culture to a plate, take ODs, then add 100 μL of Nano-Glo luciferase reagent. The Nano-Glo will lyse cells, making OD measurements taken after addition unreliable.
Figure 3. nsAA incorporation data. (A) Endpoint data for nsAA incorporation, using different nsAAs for different AARSs: BipA for bipARS, pAzF for napARS, CouAA for CouRS, BpA for bpaRS, boc-K for the AbkRS. Data processed as discussed in Data Analysis. Biological triplicates were averaged; error bars represent the standard deviation. (B) Time courses for nsAA incorporation; shaded area is standard deviation among biological triplicates.
Variant for incorporation with ammoniaS750 (slower growth, less reliable, more accessible import)
1. When trying to do incorporation of nsAAs that do not import well into the cell (likely due to being large and hydrophobic), import may be improved by using media lacking competing amino acids, such as S750 where ammonia is the sole nitrogen source (see recipes for ammoniaS750).
2. Bacillus will grow in this media and incorporation will occur, but significantly more slowly. Replace both the seed culture and experimental media with ammoniaS750, and allocate roughly 50% more growth time, especially for the seed culture, which may take 6–8 h to reach a high enough OD for seeding (minimum OD is approximately 0.2).
Variant for incorporation with LB (less reliable, higher background, hindered cellular import)
1. Replace S750 LB starter culture and experimental cultures with LB.
2. Spin down cells and resuspend with PBS prior to measuring fluorescence. Cells do not need to be spun down for luminescence.

Data analysis

Data analysis of nsAA incorporation is straightforward, and results should be interpretable from raw data without manipulation, as shown in Figure 4. However, to achieve final data, follow the steps below. Also see Supplemental Excel file 1 for an example of data processing.

Average biological replicates of fluorescence/luminescence data from the WT controls without nsAA, then subtract 90% of that value from the fluorescence/luminescence values of all samples. Since low background synthetases without nsAA tend to produce very little observable background incorporation, if the full WT background is subtracted then the nsAA fluorescence values will often be negative. This complicates graphing and downstream data analysis, such as determination of fold-induction. Since the WT fluorescence is very low, the difference between 90% and 100% is extremely small—usually smaller than the standard deviation between the three WT biological replicates. This results in background-normalized incorporation data.
Subtract plate reader background OD (from reading blank media wells; it should be constant per given plate reader) from OD values. This can be anywhere between 0.001 and 0.04 for many plate readers. This results in normalized OD data. Note any especially low OD values and consider data from those wells suspect. Most nsAAs described here should not significantly hinder growth, except for 5OHW, which was observed to decrease final OD.
Divide background-normalized incorporation data by the normalized OD data. This results in OD-normalized incorporation data. Skip this step for time course analysis.
Divide all averages by OD-normalized fluorescence/luminescence data from the TAC positive control lacking a TAG stop codon. This results in final data as a fraction of protein expression without nsAA incorporation. See Figure 4 and Supplemental Excel file 1for examples.
Use standard deviation among three biological replicates to plot error bars.

Figure 4. Data analysis example. (A) Raw fluorescence data from plate reader, with no normalizations. (B) Data processed according to data normalization, with background subtracted, OD normalized, and normalized to maximum possible expression of a reporter lacking a UAG codon.

Notes

Always include WT B. subtilis (Py79) as a negative control and a TAC-reporter positive control strain to properly normalize experiment-to-experiment signal. The TAC-reporter positive control contains the sense codon TAC, which encodes tyrosine, instead of the nonsense codon TAG at the desired site of nsAA incorporation in the reporter protein. It provides a measure of how much protein expression would be expected if the rate of nsAA incorporation was not a limiting factor and normalizes data across experiments. These strains can be obtained through BGSC (see Materials and Reagents).
Adding inducer to the starter cultures is optional but not recommended, as it does not seem to have any effect on final signal. Antibiotic in the starter and experimental cultures is also optional but is unnecessary for stably integrated constructs and can slow growth. Be sure not to add antibiotic to WT controls.
Starter cultures
1. This protocol recommends starting cultures from colonies instead of freezer stocks. It is possible to start cultures from freezer stocks, but starter cultures grown this way grow unreliably and usually take longer to reach an appropriate OD. The same is true for cultures from old plates (>2–3 days). Both of these approaches can be used if needed but ensure that cells have at least gone through two doublings before starting experimental cultures from seed cultures.
2. Higher ODs than 0.7 do work for starter cultures, but results will not be as reliable.
Protocol Variants
1. The basic 300 μL S750 plate reader endpoint incorporation protocol is extremely reliable when performed correctly. When testing a new synthetase or nsAA, execute that protocol first to demonstrate that incorporation is working before attempting larger cultures, richer media, or other protocol variants.
2. Because of Bacillus’ need for aeration, it is better to shake 96-well plates overnight at 1,000 rpm with a 3 mm orbital volume. However, robust incorporation has been observed in 96-well plates with a standard incubator’s 250 rpm overnight shaking.
3. When attempting to purify protein-containing nsAA, the Gram-positive BugBuster kit was found to be very effective for purification of protein from stationary-phase Bacillus subtilis grown overnight in S750.
Background normalization
1. In general, it is not necessary to change background subtraction during data analysis based on OD. This is because B. subtilis in S750 has relatively low autofluorescence, and after overnight growth almost all cultures will have reached saturation.
2. However, other media, such as LB, will have higher autofluorescence and potentially more variable ODs. In these cases, it may be necessary to create a linear autofluorescence normalization curve, where several different cultures of WT Py79 at different ODs are measured, then plotted, and a linear autofluorescence curve created. That curve can then be used to subtract autofluorescence based on OD.

Recipes

S750 (1 L)
100 mL of 10× S750 salts
10 mL of 100× metals
10 mL of 1 M glutamate
20 mL of 50% glucose
860 mL of ddH₂O
ammoniaS750 (1 L)
100 mL of 10× S750 salts
10 mL of 100× metals
20 mL of 10% w/v ammonia sulfate
20 mL of 50% glucose
850 mL of ddH₂O
10× S750 salts (1 L)
104.7 g of MOPS (free acid)
13.2 g of ammonia sulfate, anhydrous
6.8 g of potassium phosphate monobasic, anhydrous
To 1 L with ddH₂O
100× metals (500 mL)
100 mL of 1 M magnesium chloride
35 mL of 1 M calcium chloride
2.5 mL of 1 M manganese chloride
5 mL of 10 mM zinc chloride
25 mL of 2 mg/mL thiamine-HCl
1 mL of 1 M HCl
5 mL of 50 mM iron (III) chloride
326.5 mL of ddH₂O

Acknowledgments

This work was supported by NSF collaborative research grants MCB-2027074 (to E.C.G.) and MCB-2027092 (to A.M.K.). M.A.J. was partially supported by funding from the Department of Education – Graduate Assistance in Areas of National Need (P200A210065) and from the National Institute of General Medical Sciences of the National Institutes of Health under a Chemistry-Biology Interface Training Grant (T32GM133395).

This protocol is adapted from Stork et al. (2021).

Competing interests

The authors declare the following competing interests: A.M.K. has related financial interests in Nitro Biosciences.

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