16. Utilize the protein concentration as determined by the Bradford assay in conjunction with the prior OD 600nm measurement to either determine the dose of protein per 10
7 cells or to
determine the quantity of cells needed for a particular protein
antigen dose.
3.5 Assembly
of the Polymer
Component
1. Add amine (2-aminopropane-1,3-diol, 400 mg) and diacrylate
(neopentyl glycol diacrylate) into 2 mL DMSO at 1:1.2 molar
ratio.
2. Conduct the reaction for 5 days at 60
C with continuous
stirring at 1000 rpm.
3. Polymers are purified by dialysis which is conducted against
acetone using molecular porous membrane tubing, with an
approximate molecular weight cutoff of 3500 Da.
4. Remove acetone by evaporation under vacuum.
5. Allyl-a-D-mannopyranoside (ADM) is synthesized by dissolving 3 g of D(+)-mannose and 18 mg of p-toluenesulfonyl
chloride in allyl alcohol (20 mL) at 90
C under reflux for 24 h.
6. Purify resulting ADM product via dialysis.
7. Concentrate the resulting reaction solution by vacuum distillation at 35
C (see Note 5).
8. Dissolve the synthesized acrylate-terminated polymer in
DMSO (167 mg/mL) and react with 5 M ethylene diamine
(in DMSO) at room temperature for 24 h to form aminecapped polymer.
9. Purify amine-capped polymer by dialysis followed by evaporation under vacuum.
10. React the amine-capped polymer with ADM at a 1:2 molar
ratio in DMSO at 90
C for 24 h.
11. Purify the final polymer with dialysis followed by evaporation
under vacuum.
3.6 Hybrid Vector
Preparation
1. Start an overnight culture by inoculating a hybrid vector strain
(i.e., YWT7-hly with pCJ05, pCJ06, pCJ07, pUAB055, or
pLF) into LB media containing ampicillin and grow for 16 h
at 37
C.
2. Utilize the overnight culture to seed 10 mL (2% v/v) of fresh
LB media containing ampicillin and incubate at 37
C while
periodically measuring the optical density at 600 nm
(OD 600nm ).
3. Once the optical density reaches 0.4–0.5 (see Note 6), induce
samples with 0.1 mM isopropyl β-D-1-thiogalactopyranoside
(IPTG) at 22
C for 20 h (see Note 7).
Hybrid Biological-Biomaterial Vector
469
7 cells or to
determine the quantity of cells needed for a particular protein
antigen dose.
3.5 Assembly
of the Polymer
Component
1. Add amine (2-aminopropane-1,3-diol, 400 mg) and diacrylate
(neopentyl glycol diacrylate) into 2 mL DMSO at 1:1.2 molar
ratio.
2. Conduct the reaction for 5 days at 60
C with continuous
stirring at 1000 rpm.
3. Polymers are purified by dialysis which is conducted against
acetone using molecular porous membrane tubing, with an
approximate molecular weight cutoff of 3500 Da.
4. Remove acetone by evaporation under vacuum.
5. Allyl-a-D-mannopyranoside (ADM) is synthesized by dissolving 3 g of D(+)-mannose and 18 mg of p-toluenesulfonyl
chloride in allyl alcohol (20 mL) at 90
C under reflux for 24 h.
6. Purify resulting ADM product via dialysis.
7. Concentrate the resulting reaction solution by vacuum distillation at 35
C (see Note 5).
8. Dissolve the synthesized acrylate-terminated polymer in
DMSO (167 mg/mL) and react with 5 M ethylene diamine
(in DMSO) at room temperature for 24 h to form aminecapped polymer.
9. Purify amine-capped polymer by dialysis followed by evaporation under vacuum.
10. React the amine-capped polymer with ADM at a 1:2 molar
ratio in DMSO at 90
C for 24 h.
11. Purify the final polymer with dialysis followed by evaporation
under vacuum.
3.6 Hybrid Vector
Preparation
1. Start an overnight culture by inoculating a hybrid vector strain
(i.e., YWT7-hly with pCJ05, pCJ06, pCJ07, pUAB055, or
pLF) into LB media containing ampicillin and grow for 16 h
at 37
C.
2. Utilize the overnight culture to seed 10 mL (2% v/v) of fresh
LB media containing ampicillin and incubate at 37
C while
periodically measuring the optical density at 600 nm
(OD 600nm ).
3. Once the optical density reaches 0.4–0.5 (see Note 6), induce
samples with 0.1 mM isopropyl β-D-1-thiogalactopyranoside
(IPTG) at 22
C for 20 h (see Note 7).
Hybrid Biological-Biomaterial Vector
469