13. N,N,N,N-Tetramethylethylene-diamine (TEMED).
14. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 1.8 mM KH2PO4, pH of 7.4.
15. Paraformaldehyde (PFA): 4% aqueous solution.
2.4 Materials
for Hybrid Vector
Assembly
1. D(+)-Mannose (3 g).
2. p-Toluenesulfonyl chloride: at 900 mg/L, dissolved in allyl
alcohol.
3. Ethylene diamine: 5 M in DMSO.
4. Neopentyl glycol diacrylate.
5. 2-Aminopropane-1,3-diol.
6. NaOAc: 25 mM.
3 Methods
3.1 Isolation
of Streptococcus
pneumoniae Antigens
1. Streak a cryogenic stock of the D39 Streptococcus pneumoniae
strain onto a Todd Hewitt blood agar plate using a sterile
cue-tip.
2. Incubate the plate at 37
C for 16–20 h.
3. Collect with a sterile toothpick a small portion of a colony into
5 μL of deionized water and incubate at 98
C for 10 min to
serve as the template for colony PCR.
4. Amplify antigen genes (dexB, glpO, and stkP) by PCR with the
cloning primers (Primers 1–6) using the Phusion HF DNA
polymerase (New England Biolabs) under the following conditions: 30 s at 98
C for initial denaturation, 30 amplification
cycles (10 s at 98
C, 15 s for annealing, and 72
C for
extension at the rate of 30 s per kb), 4 min at 72
C for final
extension and holding at 4
C when finished.
5. Confirm PCR products using agarose gel (1% w/v) with 0.1%
w/v SYBR Safe DNA Gel Stain electrophoresis at a constant
voltage (100 V).
6. Visualize the PCR products with UV light to confirm bands are
the correct size, as compared to a 1 kb DNA ladder standard.
7. Extract desired bands from the gel using a razor and purify the
DNA using a Thermo Scientific Gel Extraction kit following
the manufacturer’s instructions.
3.2 Assembly
of the Individual
Pneumococcal Antigen
Plasmids
1. Prepare the PCR products for cloning into the pET21c plasmid
(Amp
R ) via digestion of 2 μg of DNA with the flanking restriction sites: NdeI and XhoI (dexB), SacI and XhoI (glpO), and
NdeI and NotI (stkP). Digestions should be carried out for 1 h
at 37
C.
Hybrid Biological-Biomaterial Vector
465
14. Phosphate buffered saline (PBS): 137 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 1.8 mM KH2PO4, pH of 7.4.
15. Paraformaldehyde (PFA): 4% aqueous solution.
2.4 Materials
for Hybrid Vector
Assembly
1. D(+)-Mannose (3 g).
2. p-Toluenesulfonyl chloride: at 900 mg/L, dissolved in allyl
alcohol.
3. Ethylene diamine: 5 M in DMSO.
4. Neopentyl glycol diacrylate.
5. 2-Aminopropane-1,3-diol.
6. NaOAc: 25 mM.
3 Methods
3.1 Isolation
of Streptococcus
pneumoniae Antigens
1. Streak a cryogenic stock of the D39 Streptococcus pneumoniae
strain onto a Todd Hewitt blood agar plate using a sterile
cue-tip.
2. Incubate the plate at 37
C for 16–20 h.
3. Collect with a sterile toothpick a small portion of a colony into
5 μL of deionized water and incubate at 98
C for 10 min to
serve as the template for colony PCR.
4. Amplify antigen genes (dexB, glpO, and stkP) by PCR with the
cloning primers (Primers 1–6) using the Phusion HF DNA
polymerase (New England Biolabs) under the following conditions: 30 s at 98
C for initial denaturation, 30 amplification
cycles (10 s at 98
C, 15 s for annealing, and 72
C for
extension at the rate of 30 s per kb), 4 min at 72
C for final
extension and holding at 4
C when finished.
5. Confirm PCR products using agarose gel (1% w/v) with 0.1%
w/v SYBR Safe DNA Gel Stain electrophoresis at a constant
voltage (100 V).
6. Visualize the PCR products with UV light to confirm bands are
the correct size, as compared to a 1 kb DNA ladder standard.
7. Extract desired bands from the gel using a razor and purify the
DNA using a Thermo Scientific Gel Extraction kit following
the manufacturer’s instructions.
3.2 Assembly
of the Individual
Pneumococcal Antigen
Plasmids
1. Prepare the PCR products for cloning into the pET21c plasmid
(Amp
R ) via digestion of 2 μg of DNA with the flanking restriction sites: NdeI and XhoI (dexB), SacI and XhoI (glpO), and
NdeI and NotI (stkP). Digestions should be carried out for 1 h
at 37
C.
Hybrid Biological-Biomaterial Vector
465