3.3 Evaluating
Membrane Disruption
by LyE
1. Conduct steps 1–4 as outlined in Subheading 3.2 (see Note 7).
2. Transfer 200 μL of culture to a 1.5 mL Eppendorf tube every
half hour postinduction over the course of 12 h.
3. Harvest cells via centrifugation at 4
C and 13,000 rpm (using
a microfuge) for 10 min.
4. Remove the supernatant and resuspend the cellular pellet in
1 mL phosphate buffered saline.
5. Lyse the cells via sonication using a Branson 450D sonifier
(400 W, tapered microtip) at 20% capacity for 5 s. Include a
sample that is not sonicated to enable determination of the
percent of cells that are disrupted.
6. Following sonication, plate the cells on LB agar plates (diluting
as needed to enable accurate single colony formation and
counting) and incubate for 24 h at 37
C.
7. Quantify cell disruption by counting colony forming units
(CFUs) with comparison to E. coli without any introduced
recombinant cellular disruption mechanism.
3.4 Quantifying
Protein and DNA
Release
1. Inoculate E. coli strains into 3 mL of lysogeny broth (LB) medium containing the appropriate antibiotics within an orbital
shaker at 250 rpm and 37
C and grow overnight for 16–20 h.
2. Transfer E. coli into 10 mL of fresh antibiotic-containing LB
medium at 37
C with 2.5% (v/v) inoculum from starter
cultures.
3. Measure cellular density (via OD 600nm measurement) every
hour until reaching an OD 600nm value of 0.4–0.5.
4. Induce gene expression through the addition of IPTG
(100 μM) and incubate the culture at 22
C for another hour.
As a positive control, Polymyxin B (PLB), a bacterial cell wall
disruption agent, should be added during IPTG induction to
the final concentration of 0.5 mg/mL (see Note 8).
5. Standardize cultures for each strain to a 0.5 OD 600nm by measuring cellular density every 15 min (see Note 9).
6. After standardization, centrifuge the bacterial cultures at 4
C
at 13,000 rpm (using a microfuge) for 10 min.
7. Transfer the supernatant to a 15 mL centrifuge tube to measure
absorbance: 260 nm for DNA and 280 nm for protein quantification with Genesys 20 plate reader (Fig. 4).
8. Save one milliliter of the supernatant and the bacterial pellet for
the hemolysis assay. Both the supernatant and pellet can be
stored at À20
C until ready.
Bactofection Enhancement with ΦX174 Gene E
9
Membrane Disruption
by LyE
1. Conduct steps 1–4 as outlined in Subheading 3.2 (see Note 7).
2. Transfer 200 μL of culture to a 1.5 mL Eppendorf tube every
half hour postinduction over the course of 12 h.
3. Harvest cells via centrifugation at 4
C and 13,000 rpm (using
a microfuge) for 10 min.
4. Remove the supernatant and resuspend the cellular pellet in
1 mL phosphate buffered saline.
5. Lyse the cells via sonication using a Branson 450D sonifier
(400 W, tapered microtip) at 20% capacity for 5 s. Include a
sample that is not sonicated to enable determination of the
percent of cells that are disrupted.
6. Following sonication, plate the cells on LB agar plates (diluting
as needed to enable accurate single colony formation and
counting) and incubate for 24 h at 37
C.
7. Quantify cell disruption by counting colony forming units
(CFUs) with comparison to E. coli without any introduced
recombinant cellular disruption mechanism.
3.4 Quantifying
Protein and DNA
Release
1. Inoculate E. coli strains into 3 mL of lysogeny broth (LB) medium containing the appropriate antibiotics within an orbital
shaker at 250 rpm and 37
C and grow overnight for 16–20 h.
2. Transfer E. coli into 10 mL of fresh antibiotic-containing LB
medium at 37
C with 2.5% (v/v) inoculum from starter
cultures.
3. Measure cellular density (via OD 600nm measurement) every
hour until reaching an OD 600nm value of 0.4–0.5.
4. Induce gene expression through the addition of IPTG
(100 μM) and incubate the culture at 22
C for another hour.
As a positive control, Polymyxin B (PLB), a bacterial cell wall
disruption agent, should be added during IPTG induction to
the final concentration of 0.5 mg/mL (see Note 8).
5. Standardize cultures for each strain to a 0.5 OD 600nm by measuring cellular density every 15 min (see Note 9).
6. After standardization, centrifuge the bacterial cultures at 4
C
at 13,000 rpm (using a microfuge) for 10 min.
7. Transfer the supernatant to a 15 mL centrifuge tube to measure
absorbance: 260 nm for DNA and 280 nm for protein quantification with Genesys 20 plate reader (Fig. 4).
8. Save one milliliter of the supernatant and the bacterial pellet for
the hemolysis assay. Both the supernatant and pellet can be
stored at À20
C until ready.
Bactofection Enhancement with ΦX174 Gene E
9