9. HPLC-PDA-MS.
10. GC-MS.
3 Methods
3.1 In Vivo Assays
1. Create a stock of E. coli cells producing different carotenoid
substrates to use for your assays. Transform competent cells of
the selected E. coli strain with each of the pAC vectors, by using
standard transformation procedure, such as electroporation
[53] or heat-shock [54]. Grow individual colonies to prepare
glycerol stocks.
2. Make competent E. coli strains engineered to accumulate different carotenoid substrates by a standard procedure (http://
cshprotocols.cshlp.org/) and keep them at À80
C.
3. Transform the competent E. coli strains engineered to accumulate different carotenoid substrates with the selected expression
vectors harboring the CCD under study (see Notes 3 and 4)
and the empty vector, which is used as a negative control of
activity.
4. Take individual colonies to initiate a preculture in a volume of
3 mL LB containing the appropriate antibiotics, and grow
overnight at 37
C.
5. Use 2.5 mL of the overnight cultures to inoculate 50 mL of LB
with final concentration of antibiotics reduced to half, in
200 mL flasks. Grow bacteria at 37
C with shaking
(180 rpm) to an OD 600 nm of 0.5, and induced with the
appropriate agent (arabinose or IPTG), depending on the
expression vector selected. Incubate the cultures with shaking
at 28
C for 6 h, or 20
C –16
C overnight in the dark (see
Note 5).
6. Transfer the content of the flask to 50 mL tubes and harvest the
E. coli cells by centrifugation. The color intensities of the
resulting bacterial pellets will be compared by visible inspection
to control bacterial cells harboring the empty expression plasmid. Changes in intensities are in many cases an indicator for
CCD activity. However, it has been also observed that the
expression of the CCD interferes with carotenoid biosynthesis,
causing color change without cleavage activity. (You always
need a product to be sure that you have a cleavage enzyme
and the right substrate.)
7. For pigment extraction for HPLC analyses, add 2.5 mL acetone to the bacterial pellet and vortex until total resuspension.
Centrifuge al 9000 Â g for 10 min and collect the liquid part. If
the pellet remain pigmented repeat again the MeOH:CHCl 3
extraction. Combine all the extracts and dry under nitrogen. In
68
Lourdes Go ´ mez-Go ´ mez et al.
10. GC-MS.
3 Methods
3.1 In Vivo Assays
1. Create a stock of E. coli cells producing different carotenoid
substrates to use for your assays. Transform competent cells of
the selected E. coli strain with each of the pAC vectors, by using
standard transformation procedure, such as electroporation
[53] or heat-shock [54]. Grow individual colonies to prepare
glycerol stocks.
2. Make competent E. coli strains engineered to accumulate different carotenoid substrates by a standard procedure (http://
cshprotocols.cshlp.org/) and keep them at À80
C.
3. Transform the competent E. coli strains engineered to accumulate different carotenoid substrates with the selected expression
vectors harboring the CCD under study (see Notes 3 and 4)
and the empty vector, which is used as a negative control of
activity.
4. Take individual colonies to initiate a preculture in a volume of
3 mL LB containing the appropriate antibiotics, and grow
overnight at 37
C.
5. Use 2.5 mL of the overnight cultures to inoculate 50 mL of LB
with final concentration of antibiotics reduced to half, in
200 mL flasks. Grow bacteria at 37
C with shaking
(180 rpm) to an OD 600 nm of 0.5, and induced with the
appropriate agent (arabinose or IPTG), depending on the
expression vector selected. Incubate the cultures with shaking
at 28
C for 6 h, or 20
C –16
C overnight in the dark (see
Note 5).
6. Transfer the content of the flask to 50 mL tubes and harvest the
E. coli cells by centrifugation. The color intensities of the
resulting bacterial pellets will be compared by visible inspection
to control bacterial cells harboring the empty expression plasmid. Changes in intensities are in many cases an indicator for
CCD activity. However, it has been also observed that the
expression of the CCD interferes with carotenoid biosynthesis,
causing color change without cleavage activity. (You always
need a product to be sure that you have a cleavage enzyme
and the right substrate.)
7. For pigment extraction for HPLC analyses, add 2.5 mL acetone to the bacterial pellet and vortex until total resuspension.
Centrifuge al 9000 Â g for 10 min and collect the liquid part. If
the pellet remain pigmented repeat again the MeOH:CHCl 3
extraction. Combine all the extracts and dry under nitrogen. In
68
Lourdes Go ´ mez-Go ´ mez et al.