3. Following digestion, purify the reaction products using agarose
gel extraction as described above.
4. Inoculate E. coli containing the pET21c plasmid backbone into
5 mL of LB broth containing ampicillin and incubate overnight
at 37
C and 225 rpm (see Note 1).
5. Purify pACYCDuet-1 DNA from E. coli cultures using a Qiagen Miniprep Kit following the manufacturer’s instructions.
6. Following purification, digest 3 μg of the plasmid backbone
with one set of restriction enzymes listed above for 1 h at
37
C.
7. After completion of the digestion reaction, purify the insertfree backbone from a 1% agarose gel using a Thermo Scientific
Gel Extraction kit following the manufacturer’s instructions.
8. Measure the DNA concentrations of the pACYCDuet-1 and
antigen PCR product digestions using a NanoDrop spectrophotometer via absorbance at 260 nm.
9. Ligate the appropriate the digested inserts into the digested
pACYCDuet-1 constructs utilizing T4 DNA Ligase. Incubate
the ligation reaction at room temperature overnight (see Note
2). Include a negative control in which no insert is added to
confirm complete digestion of the backbone plasmid.
10. Following the overnight incubation, heat-inactivate the ligation reactions by incubating in a 65
C heat bath for 10 min.
11. After heat inactivation, transform the ligation products into
competent GeneHogs (Thermofisher Scientific) E. coli (see
Note 3). Include a negative control in which sterile water is
added to the competent cells to evaluate contamination.
12. Verify that the resulting transformant contains the desired
plasmids by colony PCR and restriction digest analysis using
the appropriate primers and enzymes, respectively. Repeat
steps 6–13 until all four antigens are transformed into a single
plasmid to form the pLF plasmid.
13. Transform the pCJ05, pCJ06, pCJ07, pUAB055, and pLF
plasmids into E. coli YWT7-hly using the chemical transformation method (see Note 3).
14. Select a single colony for each plasmid and culture with shaking
at 250 rpm under 37
C before mixing with 40% glycerol at a
1:1 ratio prior to strain cryostorage at À80
C.
3.4 Determination
of Hybrid Vector
Protein Expression
1. To ensure proper level of protein consistent with that of the
hybrid vector in future studies, the quantity of antigen present
in a given dose of hybrid vector should be determined. Inoculate the bacterial cores into 5 mL of LB media containing
ampicillin and incubate overnight at 37
C.
Hybrid Biological-Biomaterial Vector
467
gel extraction as described above.
4. Inoculate E. coli containing the pET21c plasmid backbone into
5 mL of LB broth containing ampicillin and incubate overnight
at 37
C and 225 rpm (see Note 1).
5. Purify pACYCDuet-1 DNA from E. coli cultures using a Qiagen Miniprep Kit following the manufacturer’s instructions.
6. Following purification, digest 3 μg of the plasmid backbone
with one set of restriction enzymes listed above for 1 h at
37
C.
7. After completion of the digestion reaction, purify the insertfree backbone from a 1% agarose gel using a Thermo Scientific
Gel Extraction kit following the manufacturer’s instructions.
8. Measure the DNA concentrations of the pACYCDuet-1 and
antigen PCR product digestions using a NanoDrop spectrophotometer via absorbance at 260 nm.
9. Ligate the appropriate the digested inserts into the digested
pACYCDuet-1 constructs utilizing T4 DNA Ligase. Incubate
the ligation reaction at room temperature overnight (see Note
2). Include a negative control in which no insert is added to
confirm complete digestion of the backbone plasmid.
10. Following the overnight incubation, heat-inactivate the ligation reactions by incubating in a 65
C heat bath for 10 min.
11. After heat inactivation, transform the ligation products into
competent GeneHogs (Thermofisher Scientific) E. coli (see
Note 3). Include a negative control in which sterile water is
added to the competent cells to evaluate contamination.
12. Verify that the resulting transformant contains the desired
plasmids by colony PCR and restriction digest analysis using
the appropriate primers and enzymes, respectively. Repeat
steps 6–13 until all four antigens are transformed into a single
plasmid to form the pLF plasmid.
13. Transform the pCJ05, pCJ06, pCJ07, pUAB055, and pLF
plasmids into E. coli YWT7-hly using the chemical transformation method (see Note 3).
14. Select a single colony for each plasmid and culture with shaking
at 250 rpm under 37
C before mixing with 40% glycerol at a
1:1 ratio prior to strain cryostorage at À80
C.
3.4 Determination
of Hybrid Vector
Protein Expression
1. To ensure proper level of protein consistent with that of the
hybrid vector in future studies, the quantity of antigen present
in a given dose of hybrid vector should be determined. Inoculate the bacterial cores into 5 mL of LB media containing
ampicillin and incubate overnight at 37
C.
Hybrid Biological-Biomaterial Vector
467