2. Following digestion, purify the reaction products using agarose
gel extraction as described above.
3. 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).
4. Purify pET21c DNA from E. coli cultures using a Qiagen
Miniprep Kit following the manufacturer’s instructions.
5. Following purification, digest 3 μg of the plasmid backbone
with the same restriction enzymes for 1 h at 37
C.
6. After completion of the digestion reaction, purify the insertfree plasmid backbone from a 1% agarose gel using a Thermo
Scientific Gel Extraction kit following the manufacturer’s
instructions.
7. Measure the DNA concentrations of the pET21c and antigen
PCR product digestions using a NanoDrop spectrophotometer
via absorbance at 260 nm.
8. Ligate the digested insert into the digested pET21c constructs
utilizing T4 DNA Ligase. Incubate the ligation reaction at
room temperature overnight (see Note 2). Include a negative
control for each plasmid digestion in which no insert is added
to confirm complete digestion of the backbone plasmid.
9. Following the overnight incubation, heat-inactivate the ligation reactions by incubating in a 65
C heat bath for 10 min.
10. After heat inactivation, transform the ligation products into
competent GeneHogs (Thermofisher Scientific) E. coli (see
Note 3) to form the pCJ05 (dexB), pCJ06 (glpO), and
pCJ07 (stkP) plasmids. Include a negative control in which
sterile water is added to the competent cells to evaluate
contamination.
11. Verify that the resulting transformants contain the desired
plasmids by colony PCR and restriction digest analysis using
the appropriate primers and enzymes, respectively.
3.3 Assembly
of the Consolidated
Pneumococcal Antigen
Plasmid
1. Using the confirmed plasmids, as well as the pUAB055 plasmid
for pspA, as templates, amplify the dexB, glpO, stkP, and pspA
genes using the listed pLF primers (Primers 7–14) via PCR
with the Phusion HF DNA polymerase as described above.
2. Prepare the PCR products for cloning into the pLF plasmid
(based upon the pDuet plasmids from Novagen) via digestion
of 2 μg of DNA with the flanking restriction sites: BamHI and
AscI (dexB), SalI and NotI (glpO), and NdeI and MunI (stkP),
and FseI and XhoI (pspA). Digestions should be carried out for
1 h at 37
C.
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Ruiquan Qi et al.
gel extraction as described above.
3. 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).
4. Purify pET21c DNA from E. coli cultures using a Qiagen
Miniprep Kit following the manufacturer’s instructions.
5. Following purification, digest 3 μg of the plasmid backbone
with the same restriction enzymes for 1 h at 37
C.
6. After completion of the digestion reaction, purify the insertfree plasmid backbone from a 1% agarose gel using a Thermo
Scientific Gel Extraction kit following the manufacturer’s
instructions.
7. Measure the DNA concentrations of the pET21c and antigen
PCR product digestions using a NanoDrop spectrophotometer
via absorbance at 260 nm.
8. Ligate the digested insert into the digested pET21c constructs
utilizing T4 DNA Ligase. Incubate the ligation reaction at
room temperature overnight (see Note 2). Include a negative
control for each plasmid digestion in which no insert is added
to confirm complete digestion of the backbone plasmid.
9. Following the overnight incubation, heat-inactivate the ligation reactions by incubating in a 65
C heat bath for 10 min.
10. After heat inactivation, transform the ligation products into
competent GeneHogs (Thermofisher Scientific) E. coli (see
Note 3) to form the pCJ05 (dexB), pCJ06 (glpO), and
pCJ07 (stkP) plasmids. Include a negative control in which
sterile water is added to the competent cells to evaluate
contamination.
11. Verify that the resulting transformants contain the desired
plasmids by colony PCR and restriction digest analysis using
the appropriate primers and enzymes, respectively.
3.3 Assembly
of the Consolidated
Pneumococcal Antigen
Plasmid
1. Using the confirmed plasmids, as well as the pUAB055 plasmid
for pspA, as templates, amplify the dexB, glpO, stkP, and pspA
genes using the listed pLF primers (Primers 7–14) via PCR
with the Phusion HF DNA polymerase as described above.
2. Prepare the PCR products for cloning into the pLF plasmid
(based upon the pDuet plasmids from Novagen) via digestion
of 2 μg of DNA with the flanking restriction sites: BamHI and
AscI (dexB), SalI and NotI (glpO), and NdeI and MunI (stkP),
and FseI and XhoI (pspA). Digestions should be carried out for
1 h at 37
C.
466
Ruiquan Qi et al.