Homework#10

Homework Part A

Patrick Boyle’s Lecture Questions:

1. 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?

Field: stem cells

• What combinations of transcription factors generate entirely new cell types?:

To generate all possible combinations in stem cells, measuring which combinations generate new morphologies or functions.

• How do microenvironments influence cell differentiation/reprogramming?

Create dynamic microenvironments with controlled variables

• Can stem cells self-organize into functional tissues under completely synthetic conditions?

1. If you could make metric tons of any protein, what would you make and what positive impact could you have?

If I could produce metric tons of any protein, I would choose transferrin or albumin for their potential to improve synthetic meat.

• Transferrin: It transports iron in the body. Adding it to synthetic meat would enrich it helping to combat iron deficiency and making synthetic meat closer to real meat nutritionally.
• Albumin: It regulates nutrient transport. Adding albumin could improve texture, water retention as well, making synthetic meat more similar to real meat.

Homework Part B

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

Lycopene and beta-carotene are pigments from the carotenoid group with antioxidant properties. The production of these pigments in E. coli requires the addition of the following genes from Erwinia herbicola:

Lycopene:

• crtE
• crtB
• crtI
  • plamsid used: pAC-LYC

Beta-carotene

• Additional —> crtY: This modifies lycopene to produce beta-carotene.
  • plasmid used: pAC-BETA

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

Plasmids are elements used in synthetic engineering and generally contain an antibiotic resistance gene that acts as a selection marker. This allows for the selection of colonies that have been correctly transformed, that is, that have properly assimilated the plasmid. This can be verified after the transformation process by culturing the bacteria in a medium containing a specific antibiotic, which ensures that only colonies with the integrated plasmid will grow.

• in this week protocol: chloramphenicol resistance

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

Changes in culture conditions, such as the type of medium, the presence of compounds like fructose, and temperature, could significantly affect the amount of pigment produced:

• Changes in the medium used: In this case, the use of a richer, more nutritious medium like 2YT would help the bacteria develop further and grow more rapidly, thus leading to an increase in pigment production.
• Changes in the medium's components: The addition of elements like fructose would provide the cells with additional energy (an additional carbon source). This would boost the metabolic pathways responsible for pigment production.
• Changes in temperature: Temperature changes are also relevant and are linked to the proper functioning of enzymes and plasmid stability. Therefore, choosing the optimal working temperature could also lead to an increase in production.

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

The OD600 (optical density at 600 nm) is a widely used method for estimating cell concentration in a culture medium, in this case E. coli. This is done using a spectrophotometer, which measures the amount of light absorbed or blocked when passing through the sample. In other words, the higher the OD600 value, the greater the cell density and, therefore, the turbidity of the medium. In this laboratory, the OD600 allows us to understand the growth of E. coli cultures under different conditions.

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