Bio Production
Lycopene is the red pigment that gives tomatoes their color and beta-carotene pigment that makes carrots orange. Those pigment can be made by microbes that we use biosynthesis genes "CrtE", " CrtB", "Crtl" to convert diphosphate (FPP) to lycopene and "CrtY" to beta-carotene.
Lycopene is a representative molecule from the carotenoid family, and is one of the strongest antioxidants known to date. Lycopene can then be converted into a variety of carotenoids and derivatives in different organisms through modification reactions such as cyclizations, oxygenations and dehydrogenations, which makes it one of the most important intermediates in the carotenoid family. Xian Xu, et al. 2018
● This week we use pAC-LYC or pAC-LYCipi plasmid from Addgene to produce lycopene and pAC-BETAipi plasmid for beta-carotene production.
Note, the pAC-LYC plasmid contains three genes from Erwinia herbicola: CrtE, CrtI, and CrtB. The Arabidopsis thaliana idi gene is added to the pAC-LYCipi plasmid to boost lycopene production. The pAC-BETAipi plasmid produces beta-carotene through the addition of the Erwinia herbicola crtY gene. All plasmids include the gene for chloramphenicol resistance.
I. Combine 5 ml of the specified media (LB/ LB+Fructose[6g/L]/2YT) and 100 ul of E coli with the specified plasmid content. Prepare 24 new subcultures samples in total for overnight cultures in the circular rotating shaker racks.
II. Transfer 200 ul of each grown culture into an empty well on a clear-bottom 96-well plate for estimating cell growth by optical density.
III. Transfer 1400 ul of each grown culture into a new 1.5 ml Eppendorf microcentrifuge tube. Centrifuge at 13-14,000 rpm for 1 minute. Then Discard supernatant.
IV. Apply acetone to each sample's pellet. Acetone will precipitate cellular proteins and the carotenoid pigment will go into solution. Then Isolate acetone-dissolved pigment from solid cellular precipitate. Centrifuge the samples and keep the supernatant. Dilute the transferred supernatant with 250 ul of water which is to avoid acetone corrosion of the polystyrene plates in the remaining steps.
V. Measure the absorption spectrum of all wells on the full 96-well plate using the BioTek plate reader.
To preserve the pigment, transfer samples from your plate wells onto paper and let dry. Here is a demo with beta-carotene:
