Which genes, when transferred into E. coli, will induce the production of lycopene and beta-carotene, respectively?

To enable E. coli to produce lycopene, three genes are required: crtE, crtB, and crtI. These genes encode enzymes that together convert precursor molecules into lycopene through the carotenoid biosynthetic pathway. For beta-carotene production, one additional gene is needed: crtY. This gene encodes lycopene cyclase, which converts lycopene into beta-carotene by forming cyclic end groups on the molecule.

Why do the plasmids that are transferred into E. coli need to contain an antibiotic resistance gene?

Antibiotic resistance genes in plasmids serve as a selection marker. When E. coli are transformed with plasmids carrying these genes, only those cells that have successfully taken up the plasmid will survive in the presence of the corresponding antibiotic. This ensures that the bacterial culture is enriched for cells containing the plasmid, allowing for efficient propagation and maintenance of the engineered genetic material.

What outcomes might we expect to see when we vary the media, presence of fructose, and temperature conditions of the overnight cultures?

Using a richer medium such as 2YT, which contains more yeast extract and nutrients than LB, typically results in higher cell density and greater production of recombinant proteins or metabolites like lycopene and beta-carotene. The presence of fructose in the medium can enhance lycopene production because fructose metabolism can increase the availability of precursors and energy for the carotenoid pathway. Lower incubation temperatures, such as 25 to 30 degrees Celsius, can improve protein folding and reduce the formation of unwanted byproducts, which may further increase pigment yields and overall cell health.

Generally describe what OD600 measures and how it can be interpreted in this experiment.

OD600, or optical density at 600 nanometers, measures the turbidity of a bacterial culture by quantifying how much light is scattered by the cells in suspension. A higher OD600 value indicates a greater number of bacterial cells. In experiments involving pigment production, OD600 is used to estimate cell density, which can then be related to the amount of pigment produced per cell or per culture volume.

What are other experimental setups where we may be able to use acetone to separate cellular matter from a compound we intend to measure?

Acetone is commonly used to extract hydrophobic compounds from biological samples. For example, it is often used in lipid extraction from microalgae or plant tissues, where it helps to solubilize and separate lipids from proteins and other cellular components. Acetone can also be used to extract pigments, such as chlorophylls and carotenoids, from various organisms for analytical purposes.

Why might we want to engineer E. coli to produce lycopene and beta-carotene pigments when Erwinia herbicola naturally produces them?

Engineering E. coli for pigment production offers several advantages over using native producers like Erwinia herbicola. E. coli is a well-studied organism with many available genetic tools, making it easier to manipulate and optimize for high yields. It grows rapidly on inexpensive media, which is beneficial for large-scale production. Additionally, because E. coli does not naturally produce carotenoids, the purification of the desired pigments is simpler and more efficient, as there are fewer competing compounds in the extract. Finally, E. coli can utilize a wide range of carbon sources, allowing for flexibility in optimizing production conditions.