4 Notes
1. BL21 E. coli cells are best used with the expression vectors with
modified lac and T7 promoters.
2. CCD expression plasmid should contain the CCD gene insert
of interest in the correct orientation. CCDs can be expressed as
N-terminal and C-terminal fusion proteins. We routinely
express CCDs with polyhistidine tags in E coli. Verify the
plasmid constricts by DNA sequencing.
3. Agar plates are made by first combining all solid components
together into an Erlenmeyer flask. Secondly, add the appropriate amount of deionized water and mix until the solid components have dissolved. Thirdly, autoclave agar solution to
sterilize all components. After autoclaving, allow flask to cool.
Once the agar solution has cooled, add the desired type and
amount of antibiotics and mix well by swirling. Pour 20 mL of
agar solution into sterile culture plates and allow plates to
solidify.
4. Baffled flasks enhance bacterial growth due to the increased
amount of oxygen in the media from agitation created by the
ridges in the baffled flasks.
Fig. 3 Color shift as an indicator of CCD enzymatic activity. (a) Lipid extract of
3R,3R
0 -zeaxanthin DNM micelles incubated in enzyme assay buffer. (b) Lipid
extract of 3R,3R
0 -zeaxanthin DNM micelles incubated in presence of an protein
extract containing mouse BCO2. Note the visible color shift from yellow to pale
white between the control and enzyme sample, which is indicative for the
conversion of the carotenoid substrate to apocarotenoid products
84
Linda Dora Thomas et al.
1. BL21 E. coli cells are best used with the expression vectors with
modified lac and T7 promoters.
2. CCD expression plasmid should contain the CCD gene insert
of interest in the correct orientation. CCDs can be expressed as
N-terminal and C-terminal fusion proteins. We routinely
express CCDs with polyhistidine tags in E coli. Verify the
plasmid constricts by DNA sequencing.
3. Agar plates are made by first combining all solid components
together into an Erlenmeyer flask. Secondly, add the appropriate amount of deionized water and mix until the solid components have dissolved. Thirdly, autoclave agar solution to
sterilize all components. After autoclaving, allow flask to cool.
Once the agar solution has cooled, add the desired type and
amount of antibiotics and mix well by swirling. Pour 20 mL of
agar solution into sterile culture plates and allow plates to
solidify.
4. Baffled flasks enhance bacterial growth due to the increased
amount of oxygen in the media from agitation created by the
ridges in the baffled flasks.
Fig. 3 Color shift as an indicator of CCD enzymatic activity. (a) Lipid extract of
3R,3R
0 -zeaxanthin DNM micelles incubated in enzyme assay buffer. (b) Lipid
extract of 3R,3R
0 -zeaxanthin DNM micelles incubated in presence of an protein
extract containing mouse BCO2. Note the visible color shift from yellow to pale
white between the control and enzyme sample, which is indicative for the
conversion of the carotenoid substrate to apocarotenoid products
84
Linda Dora Thomas et al.