Assume that all of the molecular biology work you'd like to do could be automated, what sort of new biological questions would you ask, or what new types of products would you make?
For example, if all the different elements of a cell could be produced in vitro, could we create a combinatorial library to try and identify the bare minimum elements required for a cell to be reconstituted?
Could we create entire expression libraries of proteins, from plasmid design to purification, to find the most efficient combination of elements for the highest yield?
Could we design novel proteins and synthesise all the possible genetic combinations to produce them, creating DNA libraries that would produce different possible forms of our desired protein, and compare their expression and function?
If you could make metric tons of any protein, what would you make and what positive impact could you have?
Vegetable proteins (Soy, Maize, for example) would be a great use for a production such as this. With a well-established process, such large amounts of protein could be used to tackle food safety around the world, reduce the agricultural impact of crops, and (maybe) reduce the level of dependence on the meat industry.
Which genes when transferred into E. coli will induce the production of lycopene and beta-carotene, respectively?
Both molecules are part of the same group, the carotenoids, share common precursors (Lycopene is in fact a precursor of beta-carotene), and the genes involved in their production are all part of the same gene cluster, the crt cluster. However, they are not the same, genes in every carotenoid-producing organism.
Lycopene production can be engineered in E coli by introducing three genes from the cluster, crtE, *crtB, and crtI.* For the production of beta-carotene, an extra gene is needed, crtY, which converts lycopene into beta-carotene.
Why do the plasmids that are transferred into the E. coli need to contain an antibiotic resistance gene?
Antibiotic resistance genes are needed because they act as a selection marker for the transformed cells. That way, a media containing the specific antibiotic can be used to ensure only the transformed cells can grow in it, thus reducing the number of necessary screenings.
What outcomes might we expect to see when we vary the media, presence of fructose, and temperature conditions of the overnight cultures?
We would see varying degrees of bacterial growth. Assuming we are growing bacteria that don’t need any specific requirements, a richer media would promote higher levels of bacterial growth and thus cultures that are more turbid and with higher OD. On the opposite side, a minimal media, lower in nutrients such as amino acids, would make it harder for cells to grow. The presence of a simple sugar like fructose would also aid in bacterial growth, as long as they can process it properly. Finally, temperatures both above and below the appropriate range for the bacterial species can impact the growth, as well as protein function.
Generally describe what “OD600” measures and how it can be interpreted in this experiment.
OD600 represents the optical density of a culture at 600 nm, which is the standard measurement for the absorbance of light in a bacterial culture. The light sent through an empty culture, containing only media, would be fully absorbed and thus absorbance would be 0. Within a certain range, but not in a very precise way, a higher OD value indicates higher cell density. The more cells present, the less light makes it through the culture into the receptor, and thus the higher the optical density is.
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 useful in the extraction of cell elements with a polarity that make them difficult to [dissolve in water or lipids](https://www.laballey.com/blogs/blog/everything-you-needed-to-know-about-acetone-for-extraction#:~:text=Thanks to its intermediate polarity%2C acetone is a perfect choice,matrices of high water content.). It is extensively used for protein extraction via precipitation, but it can also be used in combination with other compounds, and can be used in DNA extraction as well.
Why might we want to engineer E. coli to produce lycopene and beta-carotene pigments when Erwinia herbicola naturally produces them?
*E. coli’*s genome is more well-known, it is easier to grow, to modify, and is much more extended in its lab use. The original bacteria is also a higher risk for plant populations or lab-grown plants, as it can be pathogenic for plants, but most importantly for humans. Thus, using a safer, more manageable species like E. coli reduces risks and makes research more efficient.