Week 12 Lab: Bioproduction

Contents

Post-lab questions (All students)

  1. Which genes when transferred into E. coli will induce the production of lycopene and beta-carotene, respectively?
    Lycopene is produced from farnesyl diphosphate with the enzymes encoded by crtE, crtB, and crtL. Then to make beta-carotene, they need the additional enzyme encoded by crtY.
  2. Why do the plasmids that are transferred into the E. coli need to contain an antibiotic resistance gene?
    The plasmids need an antibiotic resistance gene to ensure that the plasmid is retained. The plasmid is an extra metabolic cost for the cells to maintain, and culturing in antibiotics (that the plasmid has a resistance gene for) provides the pressure to keep and express the plasmid.
  3. What outcomes might we expect to see when we vary the media, presence of fructose, and temperature conditions of the overnight cultures?
    With different culturing conditions, the cells might grow slower or faster, or produce more or less of the goal compounds.
  4. Generally describe what “OD600” measures and how it can be interpreted in this experiment.
    OD600 is the measurement of optical density at 600 nm, which is generally used as a proxy for cell density because cells block light passing through the spectrophotometer. In this experiment, it can be interpreted into how well the cells grow.
  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?
    i’m not sure what this question is asking. like what other bioproduceable compounds are acetone-soluble?
  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 better studied, which means we have more genetic tools available to manipulate E. coli compared to E. herbicola and we know more about the metabolism so we might make more informed choices for metabolic engineering.

Post-lab questions (Committed Listeners)

  1. Let’s get in touch with our metabolic pathway.

    1. What are the enzymes of the carotene pathway?
      CrtE (geranylgeranyl diphosphate synthase), CrtB (phytoene synthase), CrtL (lycopene beta-cyclase), CrtY (lycopene cyclase), and CrtZ (beta-carotene hydroxylase).
    2. Within this pathway, which is the rate determining step (the step that takes the longest)? Which enzyme is responsible for this step?

  2. 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)?
      I would choose E. coli for production because it has a faster growth rate and there are known bacterial genes for this biosynthesis pathway. I’d really only ever choose S. cerevisiae if my product required eukaryotic biosynthetic enzymes.
    2. Now choose one of the enzymes and lets outline the parts of the construct for expression.
      I’ll go with CrtB.
    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)
      1. What is the function of a promoter?
        The promoter is the RNA-polymerase recognition site that indicates the polymerase that this is the start of a gene and to begin transcription here.
      2. What types of promoters do we have?
        There are constitutive, inducible, or repressible promoters of varying strengths. For E. coli, we would use bacterial or bacteriophage promoters (if the host strain contains phage polymerase).
      3. 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?
        To turn off transcription in response to a metabolite, a repressible promoter would be more useful. To increase transcription in response to a metabolite, an inducible promoter would be more useful.
      4. 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?
        I would use a T7 promoter because it is a strong constitutive promoter, but it would have to be in an E. coli strain that includes T7 polymerase, such as BL21(DE).

  3. Origin of replication of plasmid

    1. 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)
      1. What is the origin of replication?
        The origin of replication is the recognition point for DNA polymerase, for additional copies of the plasmid to be made - both for multiple copies within a single cell and for daughter cells in cell division.
      2. What types of origin of replication do we have?
        Ori’s are relaxed if they are positively regulated by RNA or stringent if they are positively regulated by proteins. An ori is high-copy, medium-copy, or low-copy depending on the balance between positive and negative regulation. For plasmids that are produced in E. coli for transfection into another organism, the plasmid would need to have both an E. coli ori and an ori for that other organism.
      3. (Extra) What are compatibility groups?
        Compatibility groups refers to classifications of ori’s that are derived from the same sequence, and thus use the same cellular machinery for replication. For the most predictable and reproducible copy-numbers, plasmids with ori’s in the same compatibility group shouldn’t be used together in the same strain.
      4. Now for the previously chosen promoter and gene what will be the best origin or replication?
        pMB1 ori is the ori in pUC plasmids, which is high-copy number, and therefore good for overexpression of an enzyme for increased biosynthesis.

  4. (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?
    For overexpression, I would use an RBS close to the consensus sequence, and probably a double terminator to prevent any leaky readthrough. Finally, I might add an operator like lacO (or fully replace my T7 promoter with an inducible promoter) to add inducible control over gene expression in case the high expression ended up producing a toxic amount of protein.

  5. (Hot! Extra points) What are aptamers and riboswitches and how can they be used for metabolic tuning or engineering in prokaryotes?
    Aptamers and riboswitches are nucleic acid sequences that can bind to themselves and/or specific molecules, which allows for adding inducible control to promoters that are not typically inducible.

  6. (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)
    I’d probably use Gibson assembly to join all these parts so I don’t need to worry about designing restriction sites, just designing primers. However, Golden Gate assembly might work better if I want to be sub out different parts later.

  7. (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!!!

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

    1. First let’s check some concepts of yeast engineering and homologous recombination this in this notes
    2. 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.
    3. 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.
    4. In yeast engineering we use DNA construction designs for making genome integration. What chromosome site could you use for integration of these and why?
    5. (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.