Materials and reagents

Biological materials

Strains

1. BL21(DE3) ∆serC (Addgene, #197656). This BL21(DE3) strain of E. coli has the serC gene knocked out to prevent biosynthesis of phosphoserine, which would compete with nhpSer for incorporation into proteins (Figure 1). This strain contains Release Factor 1 (RF1), the protein responsible for terminating translation at TAG amber codons, so truncated protein will be produced along with full-length nhpSer-protein. To avoid co-purification of truncated protein with full-length protein, C-terminal purification tags are recommended. For proteins that self-assemble into homo-multimers (dimers, trimers, etc.), purification can be challenging due to the possible co-purification of truncated forms that are incorporated as subunits in the assembly. If using N-terminal purification/solubilization tags, additional purifications steps may be needed to remove truncated protein species. See General note 1 for more discussion.

2. DH10b (Thermo Fisher, catalog number: EC0113). This strain can be used for faithful propagation of plasmids and for cloning needs when users wish to clone their genes of interest into the pRBC plasmid (see Plasmids below). Do not use for protein expression. Though we have not explicitly tested all of them, other classical cloning strains of E. coli can be used including NEB 10-beta (New England BioLabs, catalog number: C3019H), DH5α (e.g., Thermo Fisher, catalog number: 18258012), NEB 5-alpha (New England BioLabs, catalog number: C2987H), or TOP10 (Thermo Fisher, catalog number: C404010).

Plasmids

1. pERM2-nhpSer (Addgene, #201922): machinery plasmid for nhpSer incorporation, kanamycin resistance with pUC origin of replication. This plasmid expresses the same tRNA synthetase and EF-Tu used for pSer incorporation (Rogerson et al., 2015). The amber codon suppressing Sep-tRNA_CUA is v2.0 developed by Chin and colleagues to minimize mis-aminoacylation (Zhang et al., 2017). The Sep-tRNA and tRNA-synthetase are constitutively expressed via lpp and GlnS promoters, respectively. The EF-Tu is under control of an IPTG inducible tac promoter. Also constitutively expressed via an OXB20 promoter is the E. coli serB protein to further eliminate (hydrolyze) free pSer amino acid that may come from the media or produced by promiscuous transaminases.

2. pCDF-Frb-v1 (Addgene, #201923): expresses Streptomyces rubellomurinus FrbA, FrbB, FrbC, FrbD, and FrbE enzymes required for the biosynthesis of nhpSer. See Zhu et al. (2023) for a detailed description of this biosynthetic pathway, which is also summarized in Figure 2. Each Frb protein is expressed under the control of an engineered T7 transcriptional promoter variant and so all Frb enzyme are expressed by the addition of IPTG. This plasmid confers spectinomycin resistance and contains the CloDF origin of replication. This plasmid is large (~11 kb) and contains repetitive elements; while we have not observed instability of this plasmid, given its large size it is good practice to minimize propagations. We recommend that when you receive the DH10b cells with the pCDF-Frb-v1 plasmid from Addgene, grow up a few individual colonies overnight in liquid media [e.g., 2× YT media supplemented with spectinomycin at 100 μg/mL (see Recipes)] and make frozen glycerol stocks of the cells for long-term storage. Glycerol stocks can be made by mixing 600 μL of overnight culture with 400 μL of sterile 50% (v/v) glycerol. Place culture tube(s) in -80 °C freezer for storage. These stocks serve as a permanent, long-term source of plasmid, which can be prepared by inoculating cultures with cells from a glob of the frozen glycerol stock. Do not thaw the glycerol stock(s) once frozen.

3. pRBC-sfGFP wild-type (Addgene, #174075): expresses wild-type sfGFP control protein with C-terminal His6 tag, under a T7 transcriptional promoter, ampicillin resistance/p15a origin of replication. sfGFP is expressed by the addition of IPTG.

4. pRBC-sfGFP 150TAG (Addgene, #174076): same as above except the sfGFP gene contains a TAG amber stop codon at site N150. The TAG codon is used to direct the translational encoding of nhpSer. Note that this is also the same plasmid used for pSer encoding (Zhu et al., 2022); any protein cloned into this vector for pSer encoding will also work for nhpSer encoding. See General note 2 for more discussions.

5. pRBC-[x] wild type (you must create): expresses your wild-type protein of interest (POI). You can clone your POI into pRBC by removing the sfGFP gene from the pRBC-sfGFP wt plasmid by restriction digest with NdeI and XhoI enzymes and replacing it with your POI gene using standard cloning techniques (e.g., ligation, Gibson Assembly, or SLiCE). For reasons mentioned above, a C-terminal purification tag is preferred. For helpful tips on construct design, see Troubleshooting 1.

6. pRBC-[x] TAG (you must create): expresses your POI with nhpSer encoded at a TAG codon. You can clone your POI into pRBC by removing the sfGFP gene by restriction digest with NdeI and XhoI and replacing with your POI gene using standard cloning techniques (e.g., ligation, Gibson Assembly, or SLiCE). Using site-directed mutagenesis, change the codon to TAG (the amber stop codon) where you intend to encode nhpSer into your POI.

Reagents

1. Tryptone (e.g., VWR, catalog number: 97063-386)

2. Yeast Extract (e.g., VWR, catalog number: 97064-368)

3. NaCl (e.g., VWR, catalog number: 97061-274)

4. Agar (e.g., VWR, catalog number: 97064-336)

5. MgSO₄·7H₂O (e.g., VWR, catalog number: 97062-134)

6. K₂HPO₄ (potassium dibasic) (e.g., VWR, catalog number: 97062-234)

7. KH₂HPO₄ (potassium monobasic) (e.g., VWR, catalog number: BDH9268)

8. α-D-glucose (e.g., VWR, catalog number: 97061-168)

9. Glycerol (e.g., VWR, catalog number: BDH24388.320)

10. Isopropyl b-D-1-thiogalactopyranoside (IPTG) (e.g., Anatrace, catalog number: I1003)

11. Ampicillin (e.g., VWR, catalog number: 97061-442)

12. Kanamycin (e.g., VWR, catalog number: 75856-684)

13. Spectinomycin (e.g., VWR, catalog number: 89156-368)

14. Antifoam B (e.g., J.T. Baker, catalog number: B531-05)

15. Phos-tag acrylamide (e.g., VWR, catalog number: 101974-086)

16. Dry ice

17. Ethanol (e.g., Sigma, catalog number: 65348-M)

18. Acrylamide/Bio-acrylamide solution (e.g., Bio-Rad, catalog number: 1610146)

Solutions

1. Phosphate Buffered Saline (PBS) (e.g., VWR, catalog number: 75800-982)

2. LB/agar (see Recipes)

3. 2× YT media (see Recipes)

4. 50% (v/v) glycerol (see Recipes)

5. 10% (v/v) glycerol (see Recipes)

6. SOC media (see Recipes)

7. Starter culture media (see Recipes)

8. 1.1× Terrific Broth media (see Recipes)

9. 10× TB potassium phosphate buffer (see Recipes)

10. Ampicillin stock (see Recipes)

11. Kanamycin stock (see Recipes)

12. Spectinomycin stock (see Recipes)

13. 0.5 M IPTG (see Recipes)

Recipes

1. LB/agar media (0.5 L)

   Reagent	Final concentration	Quantity
   Tryptone	1% (w/v)	5 g
   Yeast extract	0.5% (w/v)	2.5 g
   NaCl	1.0% (w/v)	5 g
   Agar	1.5% (w/v)	7.5 g
   H2O	n/a	To 500 mL
   Total	n/a	500 mL

   1. After mixing reagents thoroughly, autoclave on standard liquid setting to sterilize. Note the agar will not go into solution until autoclaved.

   2. After autoclaving, gently swirl the bottle to ensure melted agar is evenly mixed.

      Notes:

      i. Store LB/agar bottle in a 55 °C oven and pour plates on an as needed basis. LB/agar can be stored in molten form for ~2 weeks if sterility is maintained.

      ii. If an oven is not available, plates can be poured with antibiotics once LB/agar is sufficiently cooled to touch. Plates can be stored at 4 °C for up to a week.




2. 2× YT media (1 L)

   Reagent	Final concentration	Quantity
   Tryptone	1.6% (w/v)	16 g
   Yeast extract	1.0% (w/v)	10 g
   NaCl	0.5% (w/v)	5 g
   H2O	n/a	To 1,000 mL
   Total	n/a	1 L

   1. After mixing reagents thoroughly, autoclave on the standard liquid setting to sterilize.

   2. After autoclaving, allow to cool to room temperature before use.




3. 50% (v/v) glycerol (500 mL)

   Reagent	Final concentration	Quantity
   Glycerol (100%)	50% (v/v)	250 mL
   H2O	n/a	250 mL
   Total	n/a	500 mL

   Mix by placing a suitable magnetic stir bar in a 500 mL graduated cylinder and add 250 mL of water to graduated cylinder. While stirring, pour pure glycerol to the 500 mL mark on graduated cylinder. Stir for 5 min and then transfer to a 0.5 L bottle. Sterilize by autoclaving on liquid setting.




4. 10% (v/v) glycerol (1 L)

   Reagent	Final concentration	Quantity
   Glycerol (100%)	10% (v/v)	100 mL
   H2O	n/a	900 mL
   Total	n/a	1 L

   Mix by placing a suitable magnetic stir bar in a 1,000 mL graduated cylinder and add 900 mL of water to graduated cylinder. While stirring, pour pure glycerol to the 1,000 mL mark on graduated cylinder. Stir for 5 min and then transfer to a 1 L bottle. Sterilize by autoclaving on liquid setting.




5. SOC media, 50 mL

   Reagent	Final concentration	Quantity
   2× YT media	n/a	49 mL
   1 M MgSO4	10 mM	0.5 mL
   40% (w/v) α-D-glucose	0.4% (w/v) or ~20 mM	0.5 mL
   Total	n/a	50 mL

   1. 1 M MgSO₄ can be made by mixing 12.3 g of MgSO₄·7H₂O in water up to 50 mL of total volume. Adjust mass of MgSO₄ if using the salt with different hydration status.

   2. 40% (w/v) α-D-glucose can be made by mixing 20 g of α-D-glucose with water up to 50 mL of total volume. Mix thoroughly until glucose is dissolved. Gentle heating in a microwave may facilitate dissolution of glucose.

   3. Sterilize MgSO₄, glucose, and 2× YT solutions individually by autoclaving. Allow each component to cool to room temperature and mix as indicated above. Maintain sterility while adding components together.

   4. It is easy to contaminate SOC. We suggest breaking this into 5 × 10 mL aliquots before use or making smaller batches. If sterility is maintained, SOC can be stored at room temperature indefinitely. It can also be stored at -20 °C but avoid repeated freeze/thaw.




6. Starter culture media: 2× YT + 0.5% (v/v) glycerol (50 mL)

   Reagent	Final concentration	Quantity
   2× YT media (sterile)	n/a	49.5 mL
   50% (v/v) glycerol (sterile)	0.5% (v/v)	0.5 mL
   Total	n/a	50 mL

   Prepare immediately before use.




7. 1.1× Terrific Broth media (0.9 L)

   Reagent	Final concentration (of 1×)	Quantity
   Tryptone	1.2% (w/v)	12 g
   Yeast extract	2.4% (w/v)	24 g
   Glycerol (50% [v/v])	0.5% (v/v)	10 mL
   H2O	n/a	To 900 mL
   Total	n/a	900 mL

   1. Sterilize by autoclaving on liquid setting.

   2. Do not mix TB phosphate buffer (below) until all solutions are cool and immediately before use.




8. 10× TB potassium phosphate buffer (1 L)

   Reagent	Final concentration	Quantity
   KH2PO4 (monobasic, anhydrous)	0.17 M	23.1 g
   K2HPO4 (dibasic, anhydrous)	0.72 M	125.4 g
   H2O	n/a	To 1 L
   Total	n/a	1 L

   1. The masses provided for the potassium phosphate salts are for anhydrous formulations. Hydrate forms of these salts can be used, but masses must be adjusted to maintain indicated molarity.

   2. Sterilize by autoclaving on liquid setting.

   3. Do not mix TB phosphate buffer to 1.1× TB media until all solutions are sterilized and cooled to room temperature, and immediately before use.




9. Ampicillin stock (10 mL)

   Reagent	Final concentration	Quantity
   Ampicillin	100 mg/mL	1 g
   H2O	n/a	To 10 mL
   Total	n/a	10 mL

   1. Sterilize by filtering with a 0.2 μm syringe-end filter.

   2. Store in 1 mL aliquots at -20 °C.




10. Kanamycin stock (10 mL)

    Reagent	Final concentration	Quantity
    Kanamycin	50 mg/mL	0.5 g
    H2O	n/a	To 10 mL
    Total	n/a	10 mL

    1. Sterilize by filtering with a 0.2 μm syringe-end filter.

    2. Store in 1 mL aliquots at -20 °C.




11. Spectinomycin stock (10 mL)

    Reagent	Final concentration	Quantity
    Spectinomycin	100 mg/mL	1 g
    H2O	n/a	To 10 mL
    Total	n/a	10 mL

    1. Sterilize by filtering with a 0.2 μm syringe-end filter.

    2. Store in 1 mL aliquots at -20 °C.

    Note: Do not confuse spectinomycin with streptomycin. These antibiotics are not interchangeable.




12. 0.5 M IPTG (10 mL)

    Reagent	Final concentration	Quantity
    IPTG	0.5 M	1.19 g
    H2O	n/a	To 10 mL
    Total	n/a	10 mL

    1. Sterilize by filtering with a 0.2 μm syringe-end filter.

    2. Store in 1 mL aliquots at -20 °C.

Laboratory supplies

1. 1.7 mL Eppendorf tubes (e.g., VWR, catalog number: 87003-294)

2. 0.6 mL Eppendorf tubes (e.g., VWR, catalog number: 89000-010)

3. 100 mm plates (e.g., VWR, catalog number: 470210-568)

4. 0.5 L centrifuge bottles (must fit available centrifuges/rotor)

5. 500 mL graduate cylinder

6. 15 mL conical tubes (e.g., VWR, catalog number: 89126-798)

7. 50 mL conical tubes (e.g., VWR, catalog number: 89039-656)

8. 14 mL sterile culture tubes (e.g., VWR, catalog number: 60818-689)

9. 250 mL baffled flasks (e.g., VWR, catalog number: 89095-266)

10. 2.8 L baffled Fernbach flasks (e.g., VWR, catalog number: 22877-168)

11. Micro pipette tips 10 μL (e.g., VWR, catalog number: 76323-394)

12. Micro pipette tips 200 μL (e.g., VWR, catalog number: 76323-390)

13. Micro pipette tips 1,000 μL (e.g., VWR, catalog number: 76323-454)

14. 0.2 μm syringe end filter (e.g., VWR, catalog number: 28145-477)

15. 10 mL syringes (e.g., VWR, catalog number: 76124-664)

Equipment

1. Autoclave (any capable of sterilizing liquid media and culturing materials at 121 °C, and of a saturated steam pressure of 15 PSI)

2. Expression equipment:

   1. Static incubator for growing LB/agar plates (set to 37 °C) (e.g., VWR, catalog number: 97025-630)

   2. Shaker incubator for growing liquid cultures (e.g., New Brunswick I26R, Eppendorf, catalog number: M1324-0004)

      i. Shaker should be able to rotate at 200–250 rpm

      ii. Refrigeration is necessary for expressions below room temperature (< 25 °C)

      iii. Shaker deck should have clamps to hold 250 mL and 2.8 L Fernbach flasks

   3. Optical density 600 nm spectrophotometer (e.g., Ultrospec 10, Biochrome, catalog number: 80-2116-30)

3. Competent cell preparation and cell harvesting

   1. Centrifuge capable of speeds of at least 5,000× g and able to hold volumes commensurate with culture sizes (e.g., Beckman Coulter, model: Allegra 25R)

   2. Centrifuge bottles that can withstand centrifugal forces and hold the volume of cultures grown for harvesting cells. The type of bottles depends on the centrifuge and rotor used

   3. Freezer (-80 °C), if cells are to be stored before protein is purified (e.g., VIP ECO ULT freezer, VWR, catalog number: 76305-596)

4. Electroporator (e.g., Eppendorf Eporator, Fisher Scientific, catalog number: E4309000027)

5. 0.1 cm electro-cuvettes (e.g., Bio-Rad, catalog number:1652089)

6. Fluorometer capable of reading sfGFP fluorescence (excitation 488 nm/emission 512 nm). Handheld fluorometers work well for routine fluorescence reads (e.g., Turner Biosystems, model: PicoFluor)

7. -20 °C freezer for storing plasmids and antibiotics (e.g., Fisher Scientific, catalog number: 10-549-264)

8. Ice machine (e.g., Fisher Scientific, catalog number: 09-540-003)

Procedure