Week 1 Lab: Pipetting

Week 12 Lab: Bioproduction

Post Lab Questions (Mandatory for All Students)

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

Lycopene synthesis in E. coli requires introducing three genes from Erwinia herbicola: crtE, crtI, and crtB, the three genes work together to redirect farnesyl pyrophosphate (FPP) into lycopene. To extend the pathway toward beta-carotene, a fourth gene, crtY, must also be present, as its enzyme performs the step that converts lycopene into beta-carotene.

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

Without a selectable marker, there is no reliable way to distinguish cells that took up the plasmid from those that did not. By including an antibiotic resistance gene, only the cells that successfully incorporated the plasmid will survive when the culture is grown on antibiotic-containing media, making selection easy and efficient.

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

Nutrient rich media would mostly lead to higher cell densities. Fructose may redirect carbon flux in ways that favor carotenoid precursor availability, reduce acetate accumulation, and support better recombinant gene expression whether this would lead to higher pigment per cell would need to be confirmed by normalizing absorbance values against OD600. Temperature changes involve a trade-off: 37°C promotes faster growth but may increase metabolic stress, while lower temperatures may slow growth but allow better enzyme function or pathway performance.

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

OD600 is a measure of optical density at 600 nanometers. When light passes through a bacterial suspension, cells scatter it, and denser cultures scatter more light, resulting in a higher absorbance reading. The 600 nm wavelength is a practical choice because it estimates cell number without overlapping significantly with most biological pigments. In this experiment, raw pigment absorbance values cannot be compared directly across conditions without accounting for how many cells are present. A tube with twice as many cells might appear more pigmented simply because there is more biomass. Dividing the pigment signal by OD600 normalizes production on a per-cell basis, making comparisons between conditions more meaningful.

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 is broadly useful whenever the target molecule is hydrophobic and can be dissolved away from polar cellular components. Examples include extracting chlorophyll or carotenoids from plant. Acetone also works as a protein precipitation agent, so it can double as a cleanup step to remove enzyme and membrane debris before measuring small organic compounds by absorbance or fluorescence.

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 rapidly, is easy to transform and culture, and is well studied that it’s associated genetic parts have been well characterized. Introducing the carotenoid pathway into E. coli isolates it from the complex environment of Erwinia, making it easier to study and optimize each step independently. E. coli is also far more receptive to changes on every iteration, allowing tweaking with every observation.

Post Lab Questions (For Committed Listeners)

1.1) What are the enzymes of the carotene pathway?

1.2) Within this pathway, which is the rate-determining step? Which enzyme is responsible?

The most likely rate-limiting step is the conversion of phytoene to lycopene, performed by CrtI. This reaction is more complex than the upstream steps: while CrtE and CrtB each carry out relatively concise reactions to build phytoene, CrtI must perform multiple sequential desaturation events, each requiring cofactors and oxidative chemistry. This multi-step nature makes it a strong candidate for the slowest point in the pathway. The pathway bottlenecks extend beyond a single enzyme, but CrtI remains the most plausible enzymatic rate-limiting step.

2.1) E. coli or S. cerevisiae — which would you choose for production?

E. coli would be my choice for this application. While Yeast has real advantages as a eukaryotic system, it is slower to grow and generally requires more elaborate engineering strategies than E. coli for a prokaryotic pathway like carotenoid biosynthesis.

2.2) Construct design for crtI expression I would design an expression construct around crtI, as it encodes phytoene desaturase and represents a key control point in lycopene accumulation.

Minimum functional design: ori → resistance marker → promoter → operator → RBS → crtI → terminator. For complete lycopene synthesis, crtE and crtB would also need to be expressed; adding crtY extends the pathway to beta-carotene.

2.3.i.1.a.i) What is the function of a promoter?

A promoter is the DNA sequence that positions RNA polymerase at the correct location and strand to begin transcription. It is the primary determinant of when, in which conditions, and at what level a gene is expressed.

2.3.i.1.a.ii) What types of promoters do we have?

2.3.i.1.a.iii) Turning transcription off vs. on in response to a metabolite — which promoter type for each?

To reduce transcription when a metabolite accumulates, a repressible promoter is the right tool. for example, the trp promoter, which is shut down when tryptophan levels rise. To increase transcription in the presence of a metabolite, an inducible promoter is appropriate, such as araBAD, where arabinose binding to AraC activates transcription, or the lac promoter, where IPTG relieves LacI-mediated repression.

2.3.i.1.a.iv) Promoter choice for crtI expression

For the same reasons outlined in section 2.2, I would use an inducible promoter, specifically pBAD/araBAD, for crtI. A constitutive strong promoter risks forcing excessive metabolic burden from the moment of transformation, before growth conditions have been optimized. An inducible promoter lets me turn expression on after sufficient biomass has accumulated and allows me to titrate crtI expression to find the level that maximizes lycopene output without impairing cell health. The minimal cassette would be: pBAD promoter → RBS → crtI → terminator.

3.1.i) What is the origin of replication?

The origin of replication is the specific DNA sequence at which plasmid replication initiates inside the host cell. Replication machinery recognizes this sequence and begins duplicating the plasmid from this point.

3.1.ii) What types of origins of replication do we have?

3.1.iii) What are compatibility groups?

Two plasmids are compatible if they can both be stably maintained in the same bacterial cell over many generations. Plasmids sharing the same replication and partitioning machinery compete for the same cellular resources, and one tends to be displaced over time, they are called incompatible.

Compatibility groups categorize plasmids by their replication systems, and choosing plasmids from different groups is essential when building strains that carry more than one construct simultaneously. For instance, pUC, pBR322 belong to group A and cannot be stably co-maintained, whereas pairing one of these with a p15A-based plasmid (group B) is generally stable.

3.1.iv) Origin of replication choice for the crtI construct

A medium-copy origin such as pBR322/pMB1 or p15A would be most appropriate. High-copy origins like pUC would amplify metabolic burden from overexpression, which is counterproductive when the goal is to optimize lycopene yield relative to cell health. If the crtI plasmid needs to coexist with another pathway plasmid for example, one carrying crtE and crtB then origins from different compatibility groups must be chosen to ensure both are stably maintained.

4. Elaboration on other bioparts for correct design and further bioproduction

Beyond the promoter and gene of interest, a functional construct needs a few other key elements. The RBS sits upstream of crtI and controls translation initiation. Stronger RBS means more protein, though I’m not yet sure how much this needs to be optimized versus just using a reliable standard sequence to start. A terminator after the coding sequence stops transcription and prevents read-through. I know this matters for construct stability, but which specific terminator to pick is something I’d need to look into more carefully.

The operator is less of a separate design decision and more of a consequence of whichever promoter system you choose, with pBAD, the AraC regulatory elements come as part of that system already.For assembly, using Gibson or Golden Gate junctions from the start makes sense because swapping parts during optimization becomes much easier later. This seems like the kind of thing that saves a lot of work down the line.

I’ve come across insulators and spacers in reading but I’m genuinely unsure when they make a meaningful difference in practice versus being more of a precaution. Including a GFP reporter in an early version of the construct seems like a reasonable way to confirm everything is working before troubleshooting the full carotenoid pathway though I suspect in a real production context people might skip straight to measuring pigment output instead.

Questions 5,6,78 were marked bonus so I skipped them. :)