Monitoring Protein Stability In Vivo Using an Intein-Based Biosensor

Materials and reagents

Biological materials

1. NEB5α E. coli (New England Biolabs, C2987H) (Table 1)

2. MC4100(DE3) [20]

Table 1. E. coli strains used in this study.

Strain number	Plasmid 1		Plasmid 2		Strain
CWL599	KanR-IntWT-MBPMUT	ApR			NEB5a
CWL600	KanR-IntWT-MBPWT	ApR			NEB5a
CWL640	KanR-IntMUT-MBPWT	ApR			NEB5a
AS917	KanR-IntWT-MBPMUT	ApR	pBAD33mut-Empty	CmR	MC4100(DE3)
AS923	KanR-IntWT-MBPMUT	ApR	pBAD33-GroEL	CmR	MC4100(DE3)
AS925	KanR-IntWT-MBPWT	ApR	pBAD33mut-Empty	CmR	MC4100(DE3)
AS931	KanR-IntWT-MBPWT	ApR	pBAD33-GroEL	CmR	MC4100(DE3)
AS933	KanR-IntWT-TagRFP675	ApR	pBAD33mut-Empty	CmR	MC4100(DE3)
AS939	KanR-IntWT-TagRFP675	ApR	pBAD33-GroEL	CmR	MC4100(DE3)

Reagents

1. Difco LB broth, Miller (BD, catalog number: 244610)

2. Difco LB agar, Miller (BD, catalog number: 244510)

3. Ampicillin sodium salt (Fisher, catalog number: BP906)

4. Kanamycin sulfate (Fisher, catalog number: BP1760)

5. Chloramphenicol (Fisher, catalog number: BP904100)

6. Ethanol (Sigma-Aldrich, catalog number: E7023)

7. L-Arabinose (Chem-IMPEX, catalog number: 29750)

8. Phosphate-buffered saline (PBS) (10×) pH 7.4, RNase-free (Fisher, catalog number: AM9625)

Solutions

1. LB medium (see Recipes)

2. LB agar (see Recipes)

3. 100 mg/mL ampicillin stock solution (1,000×) (see Recipes)

4. 50 mg/mL kanamycin stock solution (1,000×) (see Recipes)

5. 35 mg/mL chloramphenicol stock solution (1,000×) (see Recipes)

6. 20% L-arabinose stock solution (100×) (see Recipes)

Recipes

1. LB medium

25 g of LB broth

Bring to 1 L with ddH₂O

Autoclave to sterilize

2. LB agar

40 g of LB agar

Bring to 1 L with ddH₂O

Autoclave to sterilize

3. 100 mg/mL ampicillin stock solution (1,000×)

1 g of ampicillin sodium salt

Bring to 10 mL with ultrapure H₂O

Place in 10 mL syringe and pass through 0.22 mm filter to sterilize

Aliquot in 1.5 mL tubes and store at -20 °C

4. 50 mg/mL kanamycin stock solution (1,000×)

500 mg of kanamycin sulfate

Bring to 10 mL with ultrapure H₂O

Place in 10 mL syringe and pass through 0.22 mm filter to sterilize

Aliquot in 1.5 mL tubes and store at -20 °C

5. 35 mg/mL chloramphenicol stock solution (1,000×)

350 mg of chloramphenicol

Bring to 10 mL with ethanol

Place in 10 mL syringe and pass through 0.22 mm filter to sterilize

Aliquot in 1.5 mL tubes and store at -20 °C

6. 20% L-arabinose stock solution (100×)

2 g of L-arabinose

Bring to 10 mL with ultrapure H₂O

Place in 10 mL syringe and pass through 0.22 mm filter to sterilize

Aliquot in 1.5 mL tubes and store at -20 °C

Laboratory supplies

1. 1, 10, 20, 200, and 1,000 μL pipette tips (USA Scientific, Inc. TipOne, catalog numbers: 1111-3800, 1120-1810, 1111-0706, and 1111-2820)

2. 15 mL conical tubes (Greiner Bio-One, catalog number: 188 271)

3. Tissue culture plate 96 wells (Fisher Scientific, catalog number: FB012931)

4. Nonbinding 96-well plates (Greiner Bio-One, catalog number: 655904)

5. Black 96-well plates, black (Corning, catalog number: NBS3991)

6. Petri dishes (Fisher Scientific, catalog number: FB0875712)

7. 1.5 mL cuvette (Fisher Scientific, catalog number: 14955127)

8. 1.5 mL microcentrifuge tubes (USA Scientific, catalog number: 1615-5500)

9. 10 mL syringe (BD, catalog number: 302995)

10. 0.22 mm syringe filters (Fisher Scientific, catalog number: 09-719A)

11. Roll & Grow^TM spherical glass plating beads (MP Biomedicals, catalog number: 115000550)

12. Cell scraper (CELLTREAT, catalog number: 229310)

Equipment

1. Benchtop refrigerated shaking incubator (Thermo Scientific, catalog number: SHKE4000)

2. Spectrophotometer (Eppendorf, catalog number: 6131 02779)

3. Amersham Imager 600 (GE, catalog number: 29-0834-61)

4. Microplate reader (Tecan, model: Infinite M200 Pro)

5. P20 Pipetman (Gilson, catalog number: FA10003M)

6. P200 Pipetman (Gilson, catalog number: F144058M)

7. P1000 Pipetman (Gilson, catalog number: FA10006M)

8. Multichannel pipette (Thermo Scientific, catalog number: 46300100)

9. Incubator (Fisher Scientific, model: 11-690-637D)

10. Infrared thermometer (Etekcity, model: 817915020807)

11. Bunsen burner

12. 1 cm grid paper

Procedure

Procedure 1: Single expression of an intein-based biosensor

A. Prepare overnight cultures

1. In sterile conditions, prepare an overnight culture for each of the three NEB5α strains (CWL599, CWL600, and CWL640) by inoculating a culture tube that contains 5 mL of LB media and 5 μL of 1,000× ampicillin stock solution.

2. Incubate at 37 °C with shaking at 250 rpm for approximately 20 h.

B. Prepare agar plates

1. Prepare LB agar.

2. Warm LB agar until liquid, then allow it to cool to approximately 65 °C.

3. Control plates: add 1 μL of 1,000× ampicillin stock solution per 1 mL of LB agar.

4. Experimental plates: add 1 μL of 1,000× ampicillin and 1 μL of 1,000× kanamycin stock solution per 1 mL of LB agar.

5. Pour the agar into Petri dishes and allow it to solidify overnight at room temperature.

C. Cell titer standardization

1. Turn on the spectrophotometer to allow it to warm up (Figure 2).

Figure 2. Procedure for normalizing overnight cultures to OD₆₀₀ = 1. Image generated using BioRender.

2. Label two 15 mL conical tubes for each 5 mL culture as initial dilution and final dilution for each sample.

3. Fill the initial dilution tubes with 2 mL of LB broth.

4. Transfer 0.5 mL of the CWL 599 culture into its respective initial dilution tube. Pipette up and down three times to mix.

5. Repeat step C4 for the remaining samples.

6. Take 1 mL of each initial dilution mixture and transfer into its respective final dilution tube.

7. Transfer 0.9 mL of each initial dilution mixture into 2 mL cuvettes. Measure and record their OD₆₀₀ using the spectrophotometer.

8. If the OD₆₀₀ value is above 1, add LB to the final dilution tube to bring the solution to an OD₆₀₀ of 1. The equation to solve for X mL of LB added is: X = ((OD₆₀₀ Value) × 5) – 1 mL.

D. Serial dilution

1. Turn on the Bunsen burner and work near its base to prevent contamination while the nonbinding 96-well plate lid is open (Figure 3).

Figure 3. Procedure for making serial dilutions. Image generated using BioRender.

2. Assign row A to CWL 599, row B to CWL 600, and row C to CWL 640 of the nonbinding 96-well plate.

3. Using a P200 Pipetman, pipette 180 μL of LB broth into wells 2–5 in rows A–C.

4. Pipette 180 μL of each cell culture at an OD₆₀₀ value of 1 into its respective row in the empty wells in column 1. Change pipette tips between samples.

5. Using a P20 Pipetman, transfer 20 μL of sample from well A1 into well A2.

6. After drawing up the 20 μL sample, pump the plunger on the Pipetman three times to mix the 20 μL sample into the 180 μL of LB. Discard the pipette tip after mixing.

7. Transfer 20 μL of sample from well A2 into well A3.

8. After drawing up the 20 μL sample, pump the plunger on the Pipetman three times to mix the 20 μL sample into the 180 μL of LB. Discard the pipette tip after mixing.

9. Transfer 20 μL of sample from well A3 into well A4.

10. After drawing up the 20 μL sample, pump the plunger on the Pipetman three times to mix the 20 μL sample into the 180 μL of LB. Discard the pipette tip after mixing.

11. Transfer 20 μL of sample from well A4 into well A5.

12. After drawing up the 20 μL sample, pump the plunger on the Pipetman three times to mix the 20 μL sample into the 180 μL of LB. Discard the pipette tip after mixing.

13. Repeat step D11 for the remaining samples.

E. Spot titers

1. Print a 1 × 1 cm grid on paper using Excel as a template (this can be reused).

2. If plates were stored at 4 °C, warm the control and experimental agar plates in a 30 °C incubator for 10–15 min before use. If the plates were prepared the previous day, they can remain at room temperature.

3. Label plates with strain, date, and the antibiotic(s) included.

4. Arrange three control plates and three experimental plates on the grid paper.

5. Set a P10 multichannel pipette to six drops of 1.25 μL.

6. Using a P10 multichannel pipette, draw up CWL 599 samples from row A. Spot them onto each agar plate by lightly touching the tip to the surface. Avoid piercing the agar. Open the plate lid only briefly while spotting to prevent contamination. Discard pipette tips after spotting each row (Figure 4).

7. Draw up CWL 600 samples from row B and spot them below the CWL 599 spots on each agar plate. Ensure you just touch the pipette tip to the surface. Discard tips after each plate.

8. Draw up CWL 640 samples from row C and spot them below the CWL 600 spots on each agar plate. Ensure you just touch the pipette tip to the surface. Discard tips after each plate.

9. Allow the sample spots to dry at room temperature. Then, invert the plates upside-down and incubate them at 37 °C overnight (16–20 h) (Figure 5).

Note: Survival may vary depending on incubation temperature.

Figure 4. Procedure for spotting cells on agar plates. Image generated using BioRender.


Figure 5. Spot titer plate incubation and expected results. Image generated using BioRender.

Procedure 2: Dual expression of intein- or fluorescence-based biosensor with GroEL chaperone

A. Prepare overnight cultures

1. Under sterile conditions, prepare an overnight culture for each of the six MC4100 strains (AS917, AS923, AS925, AS931, AS933, and AS939) by inoculating 3 mL of LB medium with 6 μL of 1,000× ampicillin and 3 μL of 1,000× chloramphenicol stock solutions.

2. Incubate at 37 °C shaking at 250 rpm overnight (16–20 h).

B. Prepare agar plates

1. Prepare LB agar.

2. Autoclave the LB agar for 30 min at 121 °C and allow it to cool to approximately 65 °C.

3. Control plates: Per 1 mL of LB agar, add 2 μL of 1,000× ampicillin, 1 μL of 1,000× chloramphenicol, and 10 μL of 100× L-arabinose stock solution.

4. Experimental plates: Per 1 mL of LB agar, add 2 μL of 1,000× ampicillin, 1 μL of 1,000× chloramphenicol, 10 μL of 100× L-arabinose, and various concentrations of kanamycin from the 1,000× kanamycin stock solution: 0.1 μL (5 μg/mL), 0.2 μL (10 μg/mL), and 0.4 μL (20 μg/mL).

5. Non-inducing plates for GroEL chaperone: Per 1 mL of LB agar, add 2 μL of 1,000× ampicillin, 1 μL of 1,000× chloramphenicol, and various concentrations of kanamycin from the 1,000× kanamycin stock solution: 0.1 μL (5 μg/mL), 0.2 μL (10 μg/mL), and 0.4 μL (20 μg/mL).

6. Pour agar into Petri dishes and allow it to solidify overnight at room temperature.

C. Cell titer standardization

Follow the same protocol as described in Procedure 1 (nonbinding 96-well plates can be used if necessary).

D. Serial dilution

Follow the same protocol as described in Procedure 1 (nonbinding 96-well plates can be used if necessary).

E. Spot titers

1. Print a 1×1 cm grid on paper using Excel as a template (this can be reused).

2. If plates were stored at 4 °C, warm the control and experimental agar plates in a 37 °C incubator for 10–15 min before use. If the plates were prepared the previous day, they can remain at room temperature.

3. Label each plate with the strain, date, and the antibiotic(s) included.

4. Arrange three control plates and three experimental plates on the grid paper.

5. Set a P10 multichannel pipette to 5 μL.

6. Using a P10 multichannel pipette, draw up each sample (AS917, AS923, AS925, AS931, AS933, and AS939) from rows A to F.

7. Spot 5 μL of each sample onto each agar plate by lightly touching the tip to the surface. Avoid piercing the agar. Open the lid only briefly while spotting to prevent contamination. Discard pipette tips after spotting each row.

8. Allow the sample spots to dry at room temperature. Then, invert the plates upside-down and incubate them at 37 °C overnight (16–20 h) (Figure 6). Note: Survival may vary depending on incubation temperature and kanamycin concentration.

Figure 6. Spot titer plate incubation and expected results. Image generated using BioRender.

F. Fluorescence assay of TagRFP675

1. Spread 50 μL of 1.0 OD₆₀₀ AS933 and AS939 (prepared in Procedure 2, Section C) onto LB agar plates (control, experimental, and non-inducing plates) using glass plating beads (Figure 7).

2. Invert the plates upside-down and incubate them at 37 °C overnight (16–20 h).

3. Turn on the spectrophotometer and microplate reader to allow them to warm up.

4. Using a P1000 Pipetman, add 1 mL of 1× PBS (pH 7.4) to each plate and scrape the entire cell layer.

5. Transfer the cell suspension to a 1.5 mL microcentrifuge tube.

6. Centrifuge cells at 10,000× g for 1 min and discard supernatant.

7. Resuspend the cell pellet in 1 mL of 1× PBS (pH 7.4) by pipetting several times to ensure even resuspension.

8. Label two 1.5 mL microcentrifuge tubes as initial dilution and final dilution for each sample.

9. Add 900 μL of 1× PBS (pH 7.4) to each initial dilution tube.

10. Using a P200 Pipetman, transfer 100 μL of each resuspended sample into its respective initial dilution tube. Pipette up and down three times to mix.

11. Repeat step F10 for the remaining samples.

12. Transfer 1 mL of each initial dilution mixture into 2 mL cuvettes. Measure and record their OD₆₀₀ value using a spectrophotometer. If necessary, calculate the final OD₆₀₀ by multiplying by 10 (the dilution factor).

13. Take 100 μL of each initial dilution mixture and transfer it into its respective final dilution tube.

14. If OD₆₀₀ exceeds 1, adjust the sample to an OD₆₀₀ of 1 by adding 1× PBS. Use the formula: X = 0.1× (OD₆₀₀ ×10(dilution factor) −1), where X is the volume of 1× PBS (in mL) to add.

15. Using a P200 Pipetman, transfer 100 μL of each 1.0 OD₆₀₀ sample from step F14 into a black 96-well plate. For accuracy, use triplicates (i.e., three wells per sample, filling rows A–C or D–F, if needed). Change pipette tips between samples.

16. Measure and record fluorescence using a microplate reader, setting the excitation wavelength to 598 nm and the emission wavelength to 675 nm.

Figure 7. Fluorescence assay of TagRFP675. Image generated using BioRender.

Validation of protocol

This protocol has been used and validated in the following research article:

1. Son et al [14]. An intein‐based biosensor to measure protein stability in vivo. Protein Sci.

General notes and troubleshooting

1. It is critical that a second antibiotic other than kanamycin be used to maintain the intein-based folding plasmid. The backbone, pUC4K, has resistance to ampicillin, while the pBAD33 backbone has resistance to chloramphenicol, both independently of kanamycin.

2. For the intein-based folding sensor, expression of the KanR-Intein-POI fusion protein is constitutive.

3. Survival in the presence of kanamycin for cells containing the intein-based folding sensor is highly dependent on temperature, incubation time, and antibiotic concentrations. Growth also depends on whether the cells, prior to their exposure to kanamycin, were grown in liquid culture or collected from plates, as well as the growth phase of the cells.

4. The initial dilution (Figure 2) may need to be adjusted to ensure that an accurate OD_600nm reading is obtained (0.1–1.0). Additionally, based on the measured OD_600nm of a strain, the final dilution factor will need to be adjusted to obtain a titer of 10⁰.

Acknowledgments

Conceptualization, J.S.S., A.S., S.H., C.W.L.; Investigation, J.S.S., A.S., S.H., C.WL. Writing—Original Draft, J.S.S., T.M.A., A.S., S.H., C.W.L.; Writing—Review & Editing, J.S.S., T.M.A., A.S., S.H., C.W.L.; Funding acquisition, S.H. and C.W.L.; Supervision, S.H. and C.W.L. This work was supported by National Institutes of Health grants R15GM143662 and P20GM103436 (via KY INBRE) to CWL and NIH R35GM142442 to SH. This protocol is based on our previous work [14].

Competing interests

The authors declare no conflicts of interest.

References

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