Detecting Photoactivatable Cre-mediated Gene Deletion Efficiency in Escherichia coli

Materials and reagents

1. Glass test tube ϕ 16.5 × 165 mm (IWAKI, catalog number: B14-002-160)

2. Molton cap (AS ONE, catalog number: 6-352-07)

3. 1.5 mL microtube (TreffLab, catalog number: 96.07246.9.01)

4. Minisart^® syringe filter (Sartorius, catalog number: S7597-FXOSK)

5. Sterilized Petri dish ϕ90 × 15 mm (AS ONE, catalog number: 1-7484-01)

6. Aluminum foil

7. E. coli strain, carrying a fluorescently tagged target gene flanked with two loxP sequences and photoactivatable Cre (PA-Cre) system. Here, we used YK0083, which has a chloramphenicol resistance gene, cat, as a target gene for demonstration. The mCherry-tagged cat gene sandwiched with two loxP sequences was introduced into the intC locus on the genome. The genes for the PA-Cre system, creN-nmag and pmag-creC, are placed downstream of P_LlacO1 promoter on a plasmid. These genes are inducible by IPTG. This strain is available by contacting the authors.

8. Difco^TM LB broth, Miller (Luria-Bertani) (BD, catalog number: 244620)

9. Agar (Wako, catalog number: 010-15815)

10. Difco^TM M9 minimal salts, 5× (BD, catalog number: 248510)

11. D(+)-glucose (Wako, catalog number: 049-31165)

12. MgSO₄·7H₂O (Wako, catalog number: 131-00405)

13. CaCl₂·2H₂O (Wako, catalog number: 31-00435)

14. MEM amino acids solution (50×) (SIGMA, catalog number: M5550)

15. Ampicillin sodium (Wako, catalog number: 016-23301)

16. Isopropyl-β-D(-)-thiogalactopyranoside (IPTG) (Wako, catalog number: 094-05144)

17. Elix^® water produced by Milli-Q^® Integral 3 (Merck, catalog number: ZRXQ003T0)

18. Milli-Q^® water produced by Milli-Q^® Integral 3 (Merck, catalog number: ZRXQ003T0)

19. LB broth (see Recipes)

20. LB agar containing 100 μg/mL of ampicillin (Amp) (see Recipes)

21. M9 medium with 0.2% glucose and amino acids (see Recipes)

22. 1 M IPTG (see Recipes)

23. 50 mg/mL Amp (see Recipes)

Equipment

1. Centrifuge (Hitachi, himac, CT 15RE)

2. Spectrometer (Shimadzu, UV-1800)

3. Blue light illuminator (Power Supply: CCS Inc., ISC-201-2; Blue light Illuminator: CCS Inc., SLM-150X150-BB)

4. BioShaker (TAITEC, BR-21FP)

5. Incubator (MITSUBISHI ELECTRIC ENGINEERING, SLC-25A)

6. Stereomicroscope (Stereomicroscope: Olympus, SZ61; LED source: NIGHTSEA, SFA-GR)

7. Portable photodiode-based laser power meter (Gentec-EO, PRONTO-Si)

Procedure

1. Sample preparation

   1. Culture E. coli cells in LB broth with 50 μg/mL of Amp at 37 °C with shaking overnight.

   2. Centrifuge 100 μL of the overnight culture at 21,500× g for 1 min at room temperature.

   3. Remove the supernatant.

   4. Resuspend the cellular pellet in 1 mL of M9 medium with 0.2% glucose and amino acids.

   5. Measure the OD₆₀₀ of the resuspended cells.

   6. Adjust the OD₆₀₀ to 0.001 in 2 mL of M9 medium with 0.2% glucose and amino acids containing 50 μg/mL of Amp and 0.1 mM IPTG.

   7. Cover the test tube with the aluminum foil.

   8. Cultivate the cells at 37 °C with shaking for 3 h.




2. Gene deletion induction

   1. Remove the aluminum foil after the cultivation.

   2. Expose the test tube to blue light using blue-light illuminator (light intensity: 6.8 mW) for the desired duration (Figures 1 and 2, see Notes).

      
      

      
      Figure 1. Blue-light illumination inside a bio shaker with an illuminator. A blue-light illuminator was directly fastened onto a test tube spring holder inside a bio shaker. It casts a blue light downward on E. coli cultures. Light intensity was adjusted to 6.8 mW, measured with a laser power meter (λ = 470 nm).

      
      

      
      

3. Calculation of gene-deleted cells in batch

   1. Dilute the cultures exposed to blue light to OD₆₀₀ = 1.0 × 10^-6, which is equivalent to approximately 7 × 10² cells/mL.

   2. Spread 150 μL of the diluted cultures on LB agar plate containing 100 μg/mL of Amp.

   3. Cover the agar plates with aluminum foil.

   4. Incubate the plates at 37 °C for 18 h.

   5. Count the number of colonies under ambient light and excitation light for examining mCherry fluorescence using a stereomicroscope (Figure 2A).

   6. Calculate the fraction of gene-deleted cells as the number of non-fluorescent colonies divided by the number of total colonies.

Notes

The fraction of gene-deleted cells varies depending on the duration of blue-light illumination (Figure 2B). Therefore, an experimenter should determine the duration of blue-light exposure depending on the purpose of the experiment.

Cre-mediated gene deletion with blue-light illumination is detectable with loss of mCherry fluorescent signal.

Figure 2. Photoactivatable Cre-mediated gene deletion. (A) Stereomicroscopic pictures of YK0083 colonies after blue-light illumination under ambient light (left) and excitation light for detecting mCherry fluorescence (right). Cells were exposed to blue light for 1 h. Arrows show colonies generated from cells from which target genes were not deleted with blue-light illumination. (B) Blue-light illumination duration dependency of gene deletion efficiency. In case of less than 6 h blue-light illumination, test tubes were covered with aluminum foil and shaken at 37 °C, so that the time from the start of blue-light illumination to spreading on the LB agar was 6 h and consistent among the conditions. “no IPTG” indicates the condition where cells were cultured without IPTG and not exposed to blue light. Black points indicate the means. Error bars indicate standard errors (N = 239 for no IPTG, N = 327 for 0 h, N = 335 for 0.25 h, N = 373 for 0.5 h, N = 344 for 1 h, N = 350 for 2 h, N = 296 for 4 h, and N = 209 for 6 h). Reprinted/adapted from Koganezawa et al. (2022).

Recipes

1. LB broth

   1. Mix the following:

      25 g of Difco^TM LB broth, Miller (Luria-Bertani)

      1 L of Elix^® water

   2. Autoclave at 121 °C for 15 min.

   3. Store at room temperature.

   4. At the time of use, add 50 mg/mL Amp on a clean bench as appropriate, if needed.




2. LB agar containing 100 μg/mL of Amp

   1. Mix the following:

      25 g of Difco^TM LB broth, Miller (Luria-Bertani)

      15 g of agar

      1 L of Elix^® water

   2. Autoclave at 121 °C for 15 min.

   3. Add 2 mL of 50 mg/mL Amp on a clean bench.

   4. Dispense 25 mL in each 90 mm dish on a clean bench.

   5. Store at 4 °C.




3. M9 medium with 0.2% glucose and amino acids

   1. Prepare the following reagents:

      5× M9

      1) Dissolve 56.4 g of Difco^TM M9 minimal salts, 5× in 1 L of Milli-Q^® water.

      2) Autoclave at 121 °C for 15 min.

      3) Store at room temperature.

      
      

      20% glucose

      1) Dissolve 10 g of D(+)-glucose in Milli-Q^® water to 50 mL.

      2) Sterilize using a 0.2 μm pore filter.

      3) Store at 4 °C.

      
      

      1 M MgSO₄

      1) Dissolve 12.3 g of MgSO₄·7H₂O in Milli-Q^® water to 50 mL.

      2) Sterilize using a 0.2 μm pore filter.

      3) Store at 4 °C.

      
      

      1 M CaCl₂

      1) Dissolve 7.4 g of CaCl₂·2H₂O in Milli-Q^® water to 50 mL.

      2) Sterilize using a 0.2 μm pore filter.

      3) Store at 4 °C.

      
      

      Sterilized water

      1) Autoclave Milli-Q^® water at 121 °C for 15 min.

      2) Store at room temperature.

   2. Mix the following on a clean bench:

      200 mL of 5× M9

      10 mL of 20% glucose

      10 mL of MEM amino acids solution (50×)

      2 mL of 1 M MgSO₄

      100 μL of 1 M CaCl₂

      778 mL of sterilized water

   3. Store at 4 °C.

   4. At the time of use, add antibiotics or IPTG on a clean bench as appropriate, if needed.




4. 1 M IPTG

   1. Dissolve 2.383 g of IPTG in Milli-Q^® water to 10 mL.

   2. Sterilize using a 0.2 μm pore filter.

   3. Dispense in microtubes on a clean bench.

   4. Store dispensed tubes at -20 °C.




5. 50 mg/mL Amp

   1. Dissolve 500 mg of ampicillin sodium in 10 mL of Milli-Q^® water.

   2. Sterilize using a 0.2 μm pore filter.

   3. Dispense in microtubes on a clean bench.

   4. Store dispensed tubes at -20 °C.

Acknowledgments

This work was supported by JST CREST Grant Number JPMJCR1927 (Y.W.) and JPMJCR1653 (M.S.); JST ERATO Grant Number JPMJER1902 (Y.W.); Japan Society for the Promotion of Science KAKENHI Grant Number 17H06389 and 19H03216 (Y.W.); Project Grant from Kanagawa Institute of Industrial Science and Technology (KISTEC) (M. S.); and Grant-in-Aid for JSPS Fellows Grant Number JP19J22506 (Y.K.). This protocol was derived from Koganezawa et al. (2022).

Competing interests

The authors declare that no competing interests exist.

References

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