Assignment

Test the impact of culture conditions (i.e. temperature growth and growth media composition) on the production of lycopene and beta-carotene in E.coli.

Lycopene is the red pigment that gives tomatoes their color. This pigment is also made by microbes. In fact, transferring a 3-enzyme pathway to E.coli can convert farnesyl diphosphate (FPP) to lycopene. The computational tools and databases presented today can also be used to enhance lycopene production in E.coli or even produce different colors, such as the beta-carotene pigment that makes carrots orange.

For the laboratory portion of this assignment, you will characterize lycopene production in E.coli with the pAC-LYCplasmid. You will also characterize beta-carotene production in E.coli with the pAC-BETA plasmid. Note, the pAC-LYC plasmid contains three genes from Erwinia herbicola: CrtE, Crtl, and CrtB. The pAC-BETA plasmid produces beta-carotene through the addition of the Erwinia herbicola CrtY gene. All plasmids include the gene for chloramphenicol resistance.

Remember to always initial and label your tubes so you can identify their contents and distinguish them from your classmates’ material!

Step 1: Overnight Cultures

1. Setup overnight culture with conditions defined in the table below. For each condition, combine 5ml of the specified media (already supplemented with chloramphenicol) and 100ul of E.coli from the starter culture with the specified plasmid. Prepare duplicates for each condition for up to 28 new subculture samples in total.
2. Grow cultures for 20-24 hours in the circular roller drum (set to 7) within the appropriate warm room for each condition.
3. Transfer 500uL of each grown culture into an empty cuvette. We will be estimating cell growth by optical density which is measured at the wavelength of 600nm, also referred to as OD600. At 600nm, dense cell suspensions will scatter light which we can correlate to an approximate cell count. Make sure to first blank with the specified media first and then read the corresponding samples.
4. For each sample, concentrate a pellet. Fasten the culture tube’s cap tight and vortex sample as necessary!
5. Transfer 1400ul of each grown culture into a new 1.5ml Eppendorf microcentrifuge tube.
6. Centrifuge at 14,000 rpm for 1 minute.
7. Use the pipette to remove the remaining supernatant. Discard supernatant.
8. Repeat the transfer, spin, and discard twice more per sample to add onto its pellet.
9. Use the pipette to remove the remaining supernatant.