Week 12 Lab: Bioproduction
Post-lab Questions
1.Which genes when transferred into E. coli will induce the production of lycopene and beta-carotene, respectively?
crtE, crtB, and crtL produce lycopene. Adding crtY produces beta-carotene.
2.Why do the plasmids that are transferred into the E. coli need to contain an antibiotic resistance gene?
Antibiotic resistance keeps only plasmid-containing cells alive, ensuring plasmid maintenance.
3.What outcomes might we expect to see when we vary the media, presence of fructose, and temperature conditions of the overnight cultures?
Changing media, fructose, or temperature can affect cell growth and pigment production.
4.Generally describe what “OD600” measures and how it can be interpreted in this experiment.
OD600 measures cell density by light absorption at 600 nm. Higher OD600 means more cell growth.
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?
Acetone can separate pigments or other acetone-soluble compounds from cell material.
6.Why might we want to engineer E. coli to produce lycopene and beta-carotene pigments when Erwinia herbicola naturally produces them?
E. coli grows faster and is easier to genetically engineer than Erwinia herbicola.
Committed Listeners
1. What are the enzymes of the carotene pathway?
The carotene pathway enzymes are CrtE, CrtB, CrtL, CrtY, and CrtZ.
2.ithin this pathway, which is the rate determining step (the step that takes the longest)? Which enzyme is responsible for this step?
The rate-limiting step is usually phytoene synthesis, controlled by CrtB.
3.The first thing to do is to decide what organism you are going to use for this (E. coli or S. cerevisiae) for production. Which would you choose and why (emphases on production differences)?
I would choose E. coli because it grows quickly and has many genetic tools.
5.A promoter starts transcription by binding RNA polymerase.
6.Promoters can be constitutive, inducible, or repressible.
7.A repressible promoter turns genes off in response to a metabolite. An inducible promoter turns genes on.
8. I would use a T7 promoter because it gives strong gene expression in strains with T7 polymerase.
Origin of Replication
1.The origin of replication is the DNA site where plasmid copying begins.
2. Origins can be high-, medium-, or low-copy number and may be relaxed or stringent.
3. Compatibility groups classify plasmids with similar replication systems that may interfere with each other.
Additional Bioparts
1. Elaborate further on other bioparts like RBS, terminators, operators you would use for a correct design and further bioproduction?
A strong RBS improves translation, terminators stop transcription, and operators control gene expression.
2.What are aptamers and riboswitches and how can they be used for metabolic tuning or engineering in prokaryotes?
Aptamers and riboswitches regulate gene expression by binding specific molecules.
3.Now what approach can be used to join all these parts together?
Gibson assembly joins DNA fragments without restriction sites, while Golden Gate is useful for modular cloning.
4. Try to elaborate further on a biosynthetic pathway you would want to engineer in E. coli for production of a metabolite or product. What use could this bio-product have? Imagine dream applications!!!
Engineered E. coli could produce medicines, biofuels, or biodegradable plastics for industrial and medical use.(if we are imagining i would want it to be engineered shroom strains that change falvours)