Week 12: Bioproduction of Beta-Carotene and Lycopene

Post Lab Questions | Mandatory for All Students

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

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

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

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

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

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

Post Lab Questions | For Committed Listeners Only

Let’s get in touch with our metabolic pathway

  1. What are the enzymes of the carotene pathway?

  2. Within this pathway, which is the rate determining step (the step that takes the longest)? Which enzyme is responsible for this step?

Notes for design of a DNA construct for bioproduction

  1. 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)?

  2. Now choose one of the enzymes and lets outline the parts of the construct for expression

  3. For E. coli lets create a expression vector that works as a plasmid you choose E. coli let’s create a expression vector that works as a plasmids

Now, for making a functional construct there are a variety of biological parts needed for this, like ribosome binding sites, terminators, operators and promoters. The last ones are the most important in terms of enzyme or protein production. Let’s elaborate further on this biopart.

Promoter With the links below we are going to answer a few questions and think about the correct use of promoter: (https://blog.addgene.org/plasmids-101-the-promoter-region, https://www.addgene.org/mol-bio-reference/promoters/, https://blog.addgene.org/plasmids-101-repressible-promoters, https://blog.addgene.org/plasmids-101-inducible-promoters)

  • What is the function of a promoter?

  • What types of promoters do we have?

  • If we wanted to turn off the transcription of a gene in response to a metabolite, what type of promoter would be most useful? What if we wanted this to increase in the presence of the metabolite?

  • Now choose one of the genes of the metabolic pathway previously described (Carotene/lycopene )and choose one enzyme to make an expression construct. What promoter could you use for this? Why did you choose it?

Origin of replication of plasmid

With the links below we are going to answer a few questions and think about the correct use of origin of rep: (https://blog.addgene.org/plasmid-101-origin-of-replication, https://blog.addgene.org/plasmids-101-plasmid-incompatibility, https://blog.addgene.org/plasmids-101-ebook-4th-edition)

  • What is the origin of replication?

  • What types of origin of replication do we have?

  • (Extra) What are compatibility groups?

  • Now for the previously chosen promoter and gene what will be the best origin or replication?

  1. (Mandatory for Global listeners, Optional MIT/Harvard) Elaborate further on other bioparts like RBS, terminators, operators you would use for a correct design and further bioproduction?

  2. (Hot! Extra points) What are aptamers and riboswitches and how can they be used for metabolic tuning or engineering in prokaryotes?

  3. (Extra points) Now what approach can be used to join all these parts together? Make a quick analysis of their sequence in search of possibilities (search for restriction sites, etc)

  4. (Extra Hot!!! Extra Points) 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!!!

  5. (Extra points) For S. cerevisiae create an integration cassette for homologous recombination.

  • First let’s check some concepts of yeast engineering and homologous recombination this in this notes

  • As well as for prokaryotes, eukaryotic DNA designs need bioparts used for construction of a function design and further expresion. Now search for a biosynthetic pathway if interested and describe one of the genes of the pathway.

  • Now, remember that for making a functional construct there are a variety of biological parts needed for this, like ribosome binding sites or Kozak sequences, terminators, and promoters. List the ones you could use for DNA design.

  • In yeast engineering we use DNA construction designs for making genome integration. What chromosome site could you use for integration of these and why?

  • (Hot! Extra points) Following the next chart of how a DNA integration cassette should be designed and with the previously chosen parts elaborate the DNA sequence you could use to synthesize with Twist.