4. Super optimal broth with catabolite repression (SOC)
medium: 2% (w/v) peptone, 0.5% (w/v) yeast extract,
10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 ,
and 20 mM glucose.
5. Lennox broth (LB) medium: 1% (w/v) Bacto tryptone, 0.5%
(w/v) Bacto yeast extract, 0.5% (w/v) NaCl, and LB agar
plates: add 1.5 g agar-agar to 100 ml medium. Add selective
antibiotic (depending on your GGPS expression vector, e.g.,
ampicillin 150 μg ml
À1 ).
6. 1 M isopropylthiogalactoside (IPTG).
7. 50 ml plastic centrifugation tubes.
8. Centrifuge, French press.
9. GGPS Buffer A (binding buffer): 20 mM Tris–HCl, pH 8.0;
100 mM NaCl; 10 mM MgCl 2 ; 10% (v/v) glycerol.
10. GGPS Buffer B (elution buffer): GGPS Buffer A supplemented
with 100 mM imidazole.
11. TALON
® metal affinity resin, empty 5 ml column.
2.2 Recombinant
PSY
1. pCOLD plasmid containing PSY cDNA truncated by its transit
peptide.
Sufficiently high PSY expression levels allowing for purification of satisfactory amounts were obtained only when PSY
ORFs were expressed in the pCOLD cold-shock expression
system (TaKaRa). We used pCOLDI which generates
N-terminal 6ÂHis tag fusions and contains a translational
enhancer element (TEE). In contrast, high PSY expression
levels were not achieved with other expression vectors and
tags; for example, pQE, pUC, pCR2.1, pGEX, regardless of
the origin of the PSY sequence. Sequences encoding transit
peptides need to be removed, for example, after prediction by
ChloroP [28].
2. Competent Rosetta (DE3) E. coli cells.
3. LB-agar and liquid LB medium containing 100 μg ml
À1 ampicillin and 34 μg ml
À1 chloramphenicol (LB-A/C).
4. Incubator shaker, adjustable to 37
C and 15
C.
5. One 100 ml Erlenmeyer flasks with 50 ml LB-A/C each, three
1 l baffled Erlenmeyer flasks with 350 ml LB-A/C each.
6. 1 M IPTG.
7. Centrifuge and 50 ml plastic centrifugation tubes are required;
500 ml centrifugation bottles and appropriate rotor are
recommended.
8. Sonicator, French Press, ice, and overhead shaker.
Enzyme Activity of Phytoene Synthase
43
medium: 2% (w/v) peptone, 0.5% (w/v) yeast extract,
10 mM NaCl, 2.5 mM KCl, 10 mM MgCl 2 , 10 mM MgSO 4 ,
and 20 mM glucose.
5. Lennox broth (LB) medium: 1% (w/v) Bacto tryptone, 0.5%
(w/v) Bacto yeast extract, 0.5% (w/v) NaCl, and LB agar
plates: add 1.5 g agar-agar to 100 ml medium. Add selective
antibiotic (depending on your GGPS expression vector, e.g.,
ampicillin 150 μg ml
À1 ).
6. 1 M isopropylthiogalactoside (IPTG).
7. 50 ml plastic centrifugation tubes.
8. Centrifuge, French press.
9. GGPS Buffer A (binding buffer): 20 mM Tris–HCl, pH 8.0;
100 mM NaCl; 10 mM MgCl 2 ; 10% (v/v) glycerol.
10. GGPS Buffer B (elution buffer): GGPS Buffer A supplemented
with 100 mM imidazole.
11. TALON
® metal affinity resin, empty 5 ml column.
2.2 Recombinant
PSY
1. pCOLD plasmid containing PSY cDNA truncated by its transit
peptide.
Sufficiently high PSY expression levels allowing for purification of satisfactory amounts were obtained only when PSY
ORFs were expressed in the pCOLD cold-shock expression
system (TaKaRa). We used pCOLDI which generates
N-terminal 6ÂHis tag fusions and contains a translational
enhancer element (TEE). In contrast, high PSY expression
levels were not achieved with other expression vectors and
tags; for example, pQE, pUC, pCR2.1, pGEX, regardless of
the origin of the PSY sequence. Sequences encoding transit
peptides need to be removed, for example, after prediction by
ChloroP [28].
2. Competent Rosetta (DE3) E. coli cells.
3. LB-agar and liquid LB medium containing 100 μg ml
À1 ampicillin and 34 μg ml
À1 chloramphenicol (LB-A/C).
4. Incubator shaker, adjustable to 37
C and 15
C.
5. One 100 ml Erlenmeyer flasks with 50 ml LB-A/C each, three
1 l baffled Erlenmeyer flasks with 350 ml LB-A/C each.
6. 1 M IPTG.
7. Centrifuge and 50 ml plastic centrifugation tubes are required;
500 ml centrifugation bottles and appropriate rotor are
recommended.
8. Sonicator, French Press, ice, and overhead shaker.
Enzyme Activity of Phytoene Synthase
43