Week 12 HW: Building Genomes

Lab questions

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

The production of lycopene in E. coli is induced by the genes crtE, crtI, and crtB from Erwinia herbicola, which are carried in the pAC-LYC plasmid. Beta-carotene production requires the same pathway plus the additional crtY gene, present in the pAC-BETA plasmid.

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

The antibiotic resistance gene allows researchers to selectively grow only the bacteria that successfully received the plasmid. Since the media contains chloramphenicol, only transformed cells carrying the resistance gene survive and continue producing the desired pigments.

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

Changing media composition, carbon source, and temperature can affect both bacterial growth and metabolic flux toward carotenoid production. Richer media such as 2YT may increase biomass, fructose may improve recombinant gene expression and pigment synthesis, and lower temperatures like 30°C may improve folding and reduce metabolic stress, while 37°C may increase growth rate but sometimes reduce pigment accumulation.

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

OD600 measures the turbidity of the bacterial culture at 600 nm, which reflects cell density because suspended cells scatter light. In this experiment, OD600 is used to estimate bacterial growth so pigment production can be normalized relative to the number of cells present.

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 be used in experiments involving extraction of hydrophobic compounds such as pigments, lipids, chlorophylls, or carotenoids from cells or tissues. It is also useful for precipitating proteins while keeping smaller metabolites or pigments dissolved in solution for spectrophotometric analysis.

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

E. coli is easier to culture, genetically manipulate, and scale industrially than many native producers. Engineering E. coli allows researchers to optimize production conditions, tune metabolic pathways, and potentially produce carotenoids more efficiently and at lower cost for biotechnology or food applications.

What are the enzymes of the carotene pathway?

The carotene pathway described in the lab includes the enzymes encoded by crtE, crtB, crtI, and crtY. These enzymes convert metabolic intermediates such as farnesyl diphosphate into lycopene and finally into beta-carotene.

Within this pathway, which is the rate determining step? Which enzyme is responsible for this step?

A commonly limiting step in carotenoid biosynthesis is the desaturation process converting phytoene into lycopene, catalyzed by CrtI (phytoene desaturase). This step involves multiple sequential desaturation reactions and often strongly influences pathway flux.

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?

I would choose E. coli because it grows rapidly, is inexpensive to culture, and has highly developed genetic engineering tools. It is especially useful for fast and scalable bacterial bioproduction, although S. cerevisiae may be preferable for some eukaryotic proteins or pathways requiring compartmentalization.

What is the function of a promoter?

A promoter is a DNA sequence that recruits RNA polymerase and regulatory proteins to initiate transcription of a gene. It determines when, where, and how strongly a gene is expressed.

What types of promoters do we have?

Promoters can be constitutive, inducible, repressible, strong, weak, tissue-specific, or environmentally responsive. Some promoters are always active, while others respond to signals such as metabolites, temperature, or chemicals.

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 transcription off in response to a metabolite, a repressible promoter would be useful. To increase transcription in response to the metabolite, an inducible promoter would be more appropriate.

Now choose one of the genes of the metabolic pathway and choose one enzyme to make an expression construct. What promoter could you use for this? Why did you choose it?

For expression of crtI, I would use the T7 promoter in E. coli. The T7 system is widely used because it produces very high transcription levels when T7 RNA polymerase is present, which could increase carotenoid production.

What is the origin of replication?

The origin of replication (ori) is the DNA sequence where plasmid replication begins inside the host cell. It controls plasmid maintenance and copy number.

What types of origin of replication do we have?

Origins of replication can be high-copy or low-copy, broad-host-range or host-specific. Different origins determine how many plasmid copies exist per cell and which organisms can replicate the plasmid.

(Extra) What are compatibility groups?

Compatibility groups classify plasmids according to whether they can coexist in the same cell. Plasmids with similar replication control systems are usually incompatible because they interfere with each other’s replication.

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

For strong crtI expression with a T7 promoter, a medium- or high-copy-number origin such as ColE1-derived origins would be useful because it increases plasmid abundance and potentially increases enzyme production.

Elaborate further on other bioparts like RBS, terminators, operators you would use for a correct design and further bioproduction.

A strong ribosome binding site (RBS) would improve translation efficiency of the carotenoid enzymes. Terminators would ensure proper transcription termination and prevent readthrough into other genes. Operators could be added to allow metabolic regulation by repressors or activators, helping reduce metabolic burden and optimize pathway balance.

What are aptamers and riboswitches and how can they be used for metabolic tuning or engineering in prokaryotes?

Aptamers are nucleic acid sequences that bind specific molecules, while riboswitches are regulatory RNA elements that change gene expression after binding metabolites. In metabolic engineering, they can dynamically regulate enzyme expression depending on intracellular metabolite concentration, improving pathway balance and reducing toxicity.

What approach can be used to join all these parts together?

Methods such as Gibson Assembly, Golden Gate Assembly, or restriction enzyme cloning can be used to combine promoters, genes, RBSs, and terminators into a plasmid. Sequence analysis is important to avoid problematic restriction sites and ensure compatible overhangs or homologous regions for assembly.