In this lab we tested the impact temperature and growth media composition has on the production of lycopene in E coli.
Lycopene: the red pigment that gives tomatoes their color. "Bright red carotene and carotenoid pigment and phytochemical found in tomatoes and other red fruits and vegetables, such as red carrots, watermelons, etc."
Turpene: "any of a large group of volatile unsaturated hydrocarbons found in the essential oils of plants, especially conifers and citrus trees. They are based on a cyclic molecule having the formula C10H16."
Beta-barrel: "A beta barrel is a beta-sheet composed of tandem repeats that twists and coils to form a closed toroidal structure in which the first strand is bonded to the last strand (hydrogen bond)."
Chromophore: "an atom or group whose presence is responsible for the color of a compound."
The first part of the asignment consisted of mixing 24 different samples for overnight culture. The sampels were all mixed in 5mL of specified media, suplemented with chloramphenicol. 5mL is SO MUCH liquid in comparison to the previous labs. We used an electric pipette pump to suction it. The samples were as follows, 12 unique conditions, each with a duplicate. They were left overnight under the specified condition in a circular roller drum.
| Plasmid Content | Growth Media | Growth Temperature |
|---|---|---|
| pAC-LYCipi | LB | 30C |
| pAC-LYCipi | LB+Fructose(6g/L) | 30C |
| pAC-LYCipi | 2YT | 30C |
| pAC-LYCipi | 2YT+Fructose(6g/L) | 30C |
| pAC-LYCipi | LB | 37C |
| pAC-LYCipi | LB+Fructose(6g/L) | 37C |
| pAC-LYCipi | 2YT | 37C |
| pAC-LYCipi | 2YT+Fructose(6g/L) | 37C |
| pAC-LYC | LB | 37C |
| pAC-LYC | LB+Fructose(6g/L) | 37C |
| pAC-BETAipi | LB | 37C |
| pAC-BETAipi | LB+Fructose(6g/L) | 37C |
The next day a 2x200ul of each grown culture was transfered to separate wells on a clear-bottom 96-well plate to meausre growth using optical density at 600nm. Unfortunatly, I wasn't able to attend this lab, but my labmates, Rahma, Rae and Pratima, preformed the procedure and took these photos.
The overnight culture was VERY large, remember, so part of that culture was also transfered into a centrifuge tube, pelletized and suspended in acetone. Those samples were also transfered to the well plate. The second image with the pellets is taken from our lab handout.
Below shows some results from the optical density test on the different wells. The first image was the sampel plate from Noah. The second shows a single graph for one of my groups samples, and the third is the data exported into an Excel spread sheet, though that document didn't have any raw data. It is linked on the repo here.
Q. Using tools such as Biocyc and KEGG, review enzymes that can be added to (or knocked out of) your lycopene-producing E. coli to increase the amount of lycopene they produce. Compare the expected and experimental differences in production efficiency between bacteria that carry the pAC-LYC and pAC-LYCipi plasmids.
A. Looking at the Carotenoid biosynthesis pathway from KEGG it looks like there are quite a few enzymes that could be added to boost production, including phytoene, phytofluene, Z-carotene, etc.
Q. Using Biocyc and/or KEGG, can you identify the enzyme to convert the carrot-orange pigment beta-carotene to the corn-yellow pigment zeaxanthin?
A. CrtT and LUT5 convert B-carotene to B-cryptoxanthin and then to zeaxanthin
