7. Utilize the standard curve described in step 4 to determine the
cell concentration and compare to the negative control to
evaluate cytotoxicity (see Note 13).
4 Notes
1. PCR amplification is performed with Phusion DNA polymerase using the following conditions: 10 s at 98
C, 30 s for
primer annealing, 30 s per kb for extension repeated for
30 cycles.
2. Ligation reactions were carried out using 50 ng of vector DNA
and an insert to vector ratio of 5:1. The room temperature at
which the reactions were carried out was generally between
20 and 25
C.
3. Since large plasmids may be used in bactofection studies, introduction of such plasmids to E. coli cells may require use of an
alternative electro-transformation protocol instead of standard
chemical transformation.
4. This growth inhibition experiment was performed to measure
LyE expression impact upon E. coli growth. The expression
level can be adjusted by IPTG concentration and temperature.
5. At the OD 600nm listed for this step (0.4–0.5), E. coli is experiencing the maximum rate of growth during log phase and is
expected to be at peak cellular health.
6. The expected trend is that bacterial cellular viability will
decrease as LyE levels increase, due to increased IPTG induction for example.
7. The membrane disruption assessment is to measure membrane
disruption due to the LyE expression. As such, the strains were
incubated initially under the same conditions as the previous
subsection experiments.
8. Polymixin B acts as an effective antibacterial agent via cell
membrane disruption and, thus, serves as a good comparative
positive control in this case.
9. This step usually takes 1 h. However, a sufficient starting
volume should be utilized to account for the amount of liquid
removed while measuring optical density. We recommend
using an additional 10 mL beyond the amount intended to
ensure a sufficient quantity remains when an OD 600nm of 0.5 is
achieved.
10. The cell density is measured to enable seeding into individual
wells prior to transfection. Seeding levels can be adjusted as
needed to maximize final signals but one must plan accordingly
Bactofection Enhancement with ΦX174 Gene E
13
cell concentration and compare to the negative control to
evaluate cytotoxicity (see Note 13).
4 Notes
1. PCR amplification is performed with Phusion DNA polymerase using the following conditions: 10 s at 98
C, 30 s for
primer annealing, 30 s per kb for extension repeated for
30 cycles.
2. Ligation reactions were carried out using 50 ng of vector DNA
and an insert to vector ratio of 5:1. The room temperature at
which the reactions were carried out was generally between
20 and 25
C.
3. Since large plasmids may be used in bactofection studies, introduction of such plasmids to E. coli cells may require use of an
alternative electro-transformation protocol instead of standard
chemical transformation.
4. This growth inhibition experiment was performed to measure
LyE expression impact upon E. coli growth. The expression
level can be adjusted by IPTG concentration and temperature.
5. At the OD 600nm listed for this step (0.4–0.5), E. coli is experiencing the maximum rate of growth during log phase and is
expected to be at peak cellular health.
6. The expected trend is that bacterial cellular viability will
decrease as LyE levels increase, due to increased IPTG induction for example.
7. The membrane disruption assessment is to measure membrane
disruption due to the LyE expression. As such, the strains were
incubated initially under the same conditions as the previous
subsection experiments.
8. Polymixin B acts as an effective antibacterial agent via cell
membrane disruption and, thus, serves as a good comparative
positive control in this case.
9. This step usually takes 1 h. However, a sufficient starting
volume should be utilized to account for the amount of liquid
removed while measuring optical density. We recommend
using an additional 10 mL beyond the amount intended to
ensure a sufficient quantity remains when an OD 600nm of 0.5 is
achieved.
10. The cell density is measured to enable seeding into individual
wells prior to transfection. Seeding levels can be adjusted as
needed to maximize final signals but one must plan accordingly
Bactofection Enhancement with ΦX174 Gene E
13