4. Phusion DNA polymerase (New England Biolabs).
5. GeneJET Gel Extraction Kit (ThermoFisher Scientific).
6. pCMV-Luc: contains a luciferase reporter gene (Elim
Biopharmaceuticals).
7. pACYC-Duet: cloning plasmid (EMD Millipore).
8. pET29-hly: plasmid containing the listeriolysin O gene (hly)
under a T7 promoter [10].
9. MicroPulser electroporator (Bio-Rad Laboratories).
10. GENESYS 20 visible spectrophotometer (Thermo Fisher
Scientific).
11. Branson 450D sonifier (Brandson Ultrasonic Corporation).
12. Synergy 4 multi-mode microplate reader (BioTek).
Fig. 1 Mechanism of LyE lysis. The Mra Y protein is responsible for the formation of the first lipid intermediate
in cell wall biosynthesis. During lipid-1 formation, UDP-MurNac-pentapeptide attaches to the phosphate site of
C55-P through Mra Y activity with the aid of magnesium. LyE inhibits the activity of MraY and generates
immature lipids for cell wall biosynthesis resulting in the formation of transmembrane tunnels through the
bacterial cellular envelope
Bactofection Enhancement with ΦX174 Gene E
5
5. GeneJET Gel Extraction Kit (ThermoFisher Scientific).
6. pCMV-Luc: contains a luciferase reporter gene (Elim
Biopharmaceuticals).
7. pACYC-Duet: cloning plasmid (EMD Millipore).
8. pET29-hly: plasmid containing the listeriolysin O gene (hly)
under a T7 promoter [10].
9. MicroPulser electroporator (Bio-Rad Laboratories).
10. GENESYS 20 visible spectrophotometer (Thermo Fisher
Scientific).
11. Branson 450D sonifier (Brandson Ultrasonic Corporation).
12. Synergy 4 multi-mode microplate reader (BioTek).
Fig. 1 Mechanism of LyE lysis. The Mra Y protein is responsible for the formation of the first lipid intermediate
in cell wall biosynthesis. During lipid-1 formation, UDP-MurNac-pentapeptide attaches to the phosphate site of
C55-P through Mra Y activity with the aid of magnesium. LyE inhibits the activity of MraY and generates
immature lipids for cell wall biosynthesis resulting in the formation of transmembrane tunnels through the
bacterial cellular envelope
Bactofection Enhancement with ΦX174 Gene E
5