Materials and reagents

1. 15 mL centrifuge tubes (Corning, catalog number: 430791)

2. 50 mL centrifuge tubes (Corning, catalog number: 430829)

3. 500 mL triangular culture flask (Corning, catalog number: 431145)

4. 1.5 mL microtubes (Axygen, catalog number: MCT-150-C-S)

5. Sterile syringe filter (Millipore, catalog number: SLGVR33RB)

6. 500 mL centrifuge bottle (Nalgene, catalog number: 3120-0500)

7. 50 mL round bottom centrifuge tubes (Biosharp, catalog number: BS-500-MC)

8. 13.2 mL centrifuge tubes (Beckman Coulter, catalog number: 344059)

9. Escherichia coli strain EPI300 (Lucigen, catalog number: EC300110)

10. Plasmids pCNM3, pBR60, and pBBRN Pnf N50-TkTcsT, which were used previously [4,7] (Figure 1)

    
    

    
    Figure 1. Schematic illustration of the Photorhabdus virulence cassette (PVC) locus and the map of plasmids used for PVC complex and cargo protein production. A. The 16 PVC structural genes (annotated Pvc1–Pvc16, shown in blue) involved in protein complex production are encoded consecutively within the P. asymbiotica genome. B–D. The plasmids pCNM3 (B), pBR60 (C), and pBBRN Pnf N50-TkTcsT (D) were constructed by our lab and the maps were generated using SnapGene. pCNM3 was constructed by ligating the complete PVC operon into pRK404 vector for constitutive expression under their own promoters [9]. pBR60 was created by ligating the DNA fragment containing the PAU_RS16560 (LysR regulator gene, shown in blue) into pBR322 plasmid (BamHI-SalI insertion) for constitutive expression of the regulator gene under its own promoter. pBBRN Pnf N50-TkTcsT was a plasmid used for cargo production. This plasmid was created by inserting the open reading frame of the toxic TkTcsT protein (shown in blue) from Trichosanthes kirilowii into a broad host range vector pBBR1MCS5, with a PVC signal peptide sequence (shown in red) fused at the N-terminus of the TkTcsT protein. This allows the production of signal peptide–fused proteins in E. coli.

    
    

11. LB broth, powder (Solarbio, catalog number: L1010)

12. Phosphate-buffered saline (PBS) (Gibco, catalog number: C10010500BT)

13. Tetracycline HCl (Solarbio, catalog number: T8180)

14. Ampicillin, sodium salt (Solarbio, catalog number: A8180)

15. Gentamycin sulfate (Solarbio, catalog number: G8170)

16. Anhydrous ethanol (Beijing Chemical Works, catalog number: C320080)

17. TBS tablets (VWR, catalog number: K859-100TABS)

18. Triton X-100 solution (Sigma, catalog number: X-100)

19. Protease inhibitor cocktail (MCE, catalog number: HY-K0010)

20. Lysozyme (Sigma, catalog number: L6876)

21. DNase I (MCE, catalog number: HY-108882)

22. Sodium chloride (Solarbio, catalog number: S8210)

23. Magnesium chloride hexahydrate (Solarbio, catalog number: M8161)

24. Solution endotoxin erasol (TianDZ, catalog number: 60607-25)

1. LB broth medium (see Recipes)

2. 10 mg/mL tetracycline in solution (see Recipes)

3. 100 mg/mL ampicillin in solution (see Recipes)

4. 50 mg/mL gentamycin in solution (see Recipes)

5. 5 mg/mL NaCl (see Recipes)

6. 2 M MgCl₂ (see Recipes)

7. 20 mg/mL lysozyme (see Recipes)

8. 5 mg/mL DNase I (see Recipes)

9. P buffer (see Recipes)

Recipes

1. LB broth medium

   Add 25 g of LB broth powder to 1,000 mL of distilled water. Sterilize using a high-pressure steam sterilization pot at 121 °C for 15 min.




2. 10 mg/mL tetracycline in solution

   Add 0.5 g of tetracycline HCl to 50 mL of anhydrous ethanol. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.




3. 100 mg/mL ampicillin in solution

   Add 1 g of ampicillin to 10 mL of distilled water. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.




4. 50 mg/mL gentamycin in solution

   Add 1 g of gentamycin sulfate to 20 mL of distilled water. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.




5. 5 mg/mL NaCl

   Dissolve 1 g of sodium chloride in 200 mL of distilled water. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.




6. 2 M MgCl₂·6H₂O

   Dissolve 406 g of magnesium chloride hexahydrate in 800 mL of distilled water and adjust volume to 1 L. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.




7. 20 mg/mL lysozyme

   Dissolve 1 g of lysozyme in 50 mL of distilled water. Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.




8. 5 mg/mL DNase I

   Dissolve a bottle of DNase I (100 mg) in 20 mL of NaCl solution (5 mg/mL). Filter sterilize with a 0.22 µm pore size hydrophilic PVDF membrane.




9. P buffer

   1. Take 50 mL of distilled water into a 50 mL centrifuge tube and add a piece of TBS tablet. Divide the solution into two 50 mL centrifuge tubes equally after the TBS tablet is completely dissolved.

   2. Add 2.5 mL of 10% Triton X-100 solution, 500 μL of 20 mg/mL lysozyme, 500 μL of 5 mg/mL DNase I, 125 μL of 2 M MgCl₂, and 500 μL of protease inhibitor cocktail to each tube. Add distilled water to fill 50 mL.

   Notes:

   1. P buffer should be freshly prepared.

   2. TBS tablets could cause skin and eye irritation; avoid breathing dust, fumes, gas, mist, vapors, or spray.

   3. Triton X-100 solution could cause serious eye damage; wear protection when using it.

   4. The final concentration of lysozyme and DNase I are 200 µg/mL and 50 µg/mL in P buffer, respectively. Triton X-100 and MgCl₂ are 50 mg/mL, 0.5%, and 5 mM, respectively.

Equipment

1. Innova44 Incubator Shaker Series (NBS, model: SHA1104)

2. Biological safety cabinet (Shanghai Lishen Scientific Equipment Co, model: Hfsafe 1200LC)

3. Milli-Q (Millipore, catalog number: IX 70XX)

4. High-pressure steam sterilization pot (YAMATO, model: SM830)

5. High-speed refrigerated centrifuge (Eppendorf, model: 5424 R)

6. Ultracentrifuge (Beckman, model: Optima L-100 XP)

7. -80 °C ultra-low temperature freezer (Haier, model: DW-86L728J)

8. Thermostatic water bath (GFL, model: GFL1002)

9. High-speed refrigerated centrifuge (Hitachi, model: CR22G)

10. Ice machine (Panasonic, model: SIM-F140BDL)

11. NanoDrop 1000 spectrophotometer

Procedure