Cesium chloride

Chloroform

D A P I (4,,6-diamidine-2-phenylindole H C l

DNaseI (Sigma-Aldrich)

H-SM Buffer (contains l O m m o l / L H E P E S ^ N a O H solution, l O m m o l / L M g S O 4 solution, l O O m m o l / L NaCl solution, p H 7.5)

L E 3 9 2 Cells

Fluorokinase Test (P r o m e ga)

Lysogenic Esc/ieric/iia coZi, human D 1180-GFP, or D 1180 Brilliant Fluorophore Enzyme

Culture base

DMEM medium, containing 10 % fetal bovine serum, is used for the culture of COS^l, 293, A 431, NIH-3T3 cells.

MEM culture medium containing 10 % fetal bovine serum for cell culture of WI 38/VA 13/2RA, HELLA

LB Culture Base, containing 10 g/L pancreatic peptone (Becton Dickinson), 5 g/L fermented yeast extract (Becton Dickinson), 10 g/L NACL, 10 mMoL/L MgSO4, 10ug/ml ampicillin.

Phage Chromogen D N A (10n g, purified, 2. 5 X l O ⁸ copies)

Plasmids: pTrc-D, pTrc-T A T -D, pTrc-R G I > D, pTrc-V N -D, pTrc-N L S --D, constructed from pTDrcHis A (Invitrogen).

Phage

Different types of recombinant phages (2.5X ^108pfu )

TAT phage (2.5 X ^106pfu or 2.5 X ^109pfu )

Wild-type phage (2.5 X ^1010pfu )

Instrumentation

Dialysis Bags

fluorescence microscope

G F P A cube and W U cube (Olympus Optical C o .L t d , T o k y o , Japan)

class chamber slide

Incubators at 32°C, 37°C, 38°C and 45°C

2 4-well plates

Method

Preparation of recombinant X phage

Plasmid-transformed lysogenic E. coli were incubated with LB (containing 10 m m o l /L M g S O 4, 100/ng/m L 麵节西林) at 1.32°C until an absorbance value of 0.3 was reached at OD m d .

2. Incubate the bacteria at 45°C for 15 m i n , then at 38°C for 180 m i n .

3 . Treat the bacteria with bromide (final concentration 10 %) and D NASE I (final concentration IOlUgAnl) and harvest the phage particles.

4 - Two rounds of cesium chloride equilibrium centrifugation were used to isolate the phage pellets, which were then purified by dialysis in H-SM buffer.

5. Using LE392 cells as host cells, the titer was determined at 37°C by Plaqueassay.

Induction of marker gene expression with recombinant phage

6- The cells to be transfected were inoculated in 24-well plates at a density of 2.5X ¹⁰⁴ cells per well and incubated for 2 h. The cells were then incubated for 2 h in a 24-well plate.

7- Wash the cells once with medium and incubate the cells with 500ul of medium containing 2.5 X ^IO8P fu recombinant phage or IOng purified phage chromosomal DNA for 6 hours at 37°C.

8- Wash the cells twice with medium and continue incubation for 48 h.

9- Cells were collected and quantitatively analyzed for fluorokinase activity using a fluorokinase assay kit, and the results were expressed as mean relative fluorescence units (RLU) with standard deviation.

In situ detection of T A T -phage-mediated G FP expression in cultured cells

10. COS^l cells were inoculated in glass chamber slides at a density of 2. ^5XIO4 cells per well and cultured for 12 h. The cells were then incubated in a glass chamber slide for 12 h. The cells were then incubated in a glass chamber slide for 12 h.

11. Wash the cells once with medium, and then incubate the cells with 500ul of medium containing 2.5 X ^IO10pfu or 2.5 X ^109pfu of TAT phage or 2.5 X ^1010pfu of wild-type phage for 6 hours at 37°C.

12. Wash the cells twice with the medium and continue incubation for 48 h.

1 3 . The cells were collected, the nuclei were stained with D A P I and the expression of G F P was observed by fluorescence microscopy.

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