Week 12 HW: Building Genomes

Week 12 — Building Genomes

Overview

This week focused on building genomes, metabolic engineering, and biological production of valuable compounds using engineered organisms.

The lab component focused on the bioproduction of lycopene and beta-carotene in genetically modified E. coli. These carotenoid pigments are naturally associated with tomatoes and carrots, but they can also be produced in microbes by introducing the appropriate biosynthetic pathway genes.

In the lab protocol, E. coli strains carrying the plasmids pAC-LYC and pAC-BETA are used to produce lycopene and beta-carotene, respectively. The goal is to compare how different culture conditions affect bacterial growth and pigment production.

Because I was not able to complete the wet-lab experiment or collect my own absorbance data, this documentation focuses on:

1. understanding the experimental design,
2. explaining the biological logic of carotenoid bioproduction,
3. describing how the data would be analyzed,
4. answering the post-lab and Committed Listener questions,
5. and connecting CRISPR-based metabolic engineering to my final project.

Lab Overview — Bioproduction of Lycopene and Beta-Carotene

The lab uses engineered E. coli to produce two carotenoid pigments:

The plasmid pAC-LYC contains the genes crtE, crtI, and crtB from Erwinia herbicola. These genes allow E. coli to convert native isoprenoid precursors into lycopene.

The plasmid pAC-BETA contains the lycopene pathway plus crtY, which converts lycopene into beta-carotene.

The central biological challenge is that engineered cells must balance two competing goals:

1. growth, which requires cellular resources for biomass production;
2. bioproduction, which diverts metabolic flux toward the target pigment.

This is why the experiment compares different media, carbon sources, and temperatures.

Carotenoid Pathway

The simplified carotenoid pathway used in this experiment is:

FPP → GGPP → phytoene → lycopene → beta-carotene
      crtE     crtB        crtI        crtY

Therefore:

pAC-LYC = crtE + crtB + crtI → lycopene
pAC-BETA = crtE + crtB + crtI + crtY → beta-carotene

Experimental Design

The experiment compares carotenoid production across different combinations of:

The full experiment includes 16 unique culture conditions, each tested in duplicate, plus media-only controls.

Culture conditions

The goal is to determine which condition gives the highest pigment production per unit of bacterial growth.

Measurements

The lab uses two main measurements:

OD600

OD600 measures the optical density of the bacterial culture at 600 nm. It is not a direct cell count, but it estimates how much light is scattered by the bacterial suspension. A higher OD600 usually indicates more bacterial biomass.

In this experiment, OD600 is used to normalize pigment production. This is important because a culture may produce a high total amount of pigment simply because it grew more, not because each cell produced more pigment.

Pigment absorbance

After growth, the cells are pelleted and carotenoids are extracted using acetone. The extracted pigment is then measured by absorbance.

The relevant wavelengths are:

The pigment signal is then normalized by OD600:

Normalized pigment production = pigment absorbance / OD600

This gives an estimate of pigment production per unit of biomass.

Expected Analysis

If experimental data were available, I would analyze it as follows:

1. Record OD600 for each culture.
2. Extract carotenoids with acetone.
3. Measure absorbance at the pigment-specific wavelength.
4. Normalize pigment absorbance by OD600.
5. Compare normalized production across all media, carbon source, temperature, and plasmid conditions.
6. Plot pigment production per OD600 for each condition.

Example analysis table

Since I did not collect experimental measurements, I did not calculate a real best-performing condition. However, based on the experimental logic, the best condition would be the one that maximizes:

pigment absorbance / OD600

rather than pigment absorbance alone.

Post-Lab Questions — Mandatory for All Students

1. Which genes transferred into E. coli induce production of lycopene and beta-carotene?

Lycopene production requires the introduction of the carotenoid biosynthesis genes crtE, crtB, and crtI. These genes convert native isoprenoid intermediates into lycopene.

Beta-carotene production requires the lycopene pathway plus crtY. The enzyme CrtY cyclizes lycopene to form beta-carotene.

Therefore:

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

The antibiotic resistance gene allows selection of bacteria that successfully maintain the plasmid.

In this experiment, the plasmids contain an antibiotic resistance marker, such as chloramphenicol resistance. When bacteria are grown in medium containing that antibiotic, only cells carrying the plasmid can survive and grow. This is important because cells without the plasmid would not produce the carotenoid pathway enzymes and would confound the experiment.

The antibiotic resistance gene therefore helps maintain selective pressure and ensures that pigment production is linked to plasmid-containing cells.

3. What outcomes might we expect when varying media, fructose, and temperature?

Changing the medium, carbon source, and temperature can strongly affect both growth and pigment production.

Medium: Richer media such as 2YT may support more biomass than LB because they contain more nutrients. However, more growth does not always mean more pigment per cell.

Fructose: Adding fructose may improve biomass yield and metabolic flux through central carbon metabolism. This could increase precursor availability for carotenoid biosynthesis.

Temperature: Lower temperature, such as 30 °C, may reduce protein misfolding and metabolic stress, potentially improving pathway enzyme function. Higher temperature, such as 37 °C, may increase growth rate but could also increase stress or reduce pathway efficiency.

Overall, the best condition is not necessarily the one with the highest OD600. It is the one with the highest normalized pigment production.

4. What does OD600 measure and how can it be interpreted in this experiment?

OD600 measures the turbidity of a bacterial culture at 600 nm. As bacterial density increases, more light is scattered, resulting in a higher OD600 value.

In this experiment, OD600 is used as a proxy for bacterial biomass. It allows pigment production to be normalized by cell density.

For example:

High pigment absorbance + high OD600 = high total pigment, but not necessarily high production per cell
High pigment absorbance + low/moderate OD600 = potentially efficient pigment production per cell
Low pigment absorbance + high OD600 = good growth but poor bioproduction

Thus, OD600 helps distinguish between improved growth and improved metabolic production.

5. What are other experimental setups where acetone could be used to separate cellular matter from a compound we intend to measure?

Acetone can be useful when the target compound is hydrophobic or pigment-like and can be extracted away from cellular debris.

Examples include:

1. extraction of carotenoids from bacteria, yeast, algae, or plant tissues;
2. extraction of chlorophylls and other photosynthetic pigments from plant or algal samples;
3. extraction of hydrophobic secondary metabolites;
4. extraction of lipid-soluble dyes or pigments;
5. preparation of samples where proteins need to be precipitated while small hydrophobic molecules remain in solution.

In this lab, acetone disrupts cells and precipitates proteins, allowing carotenoid pigments to move into the solvent phase.

6. Why engineer E. coli to produce lycopene and beta-carotene if Erwinia herbicola naturally produces them?

There are several reasons to engineer E. coli instead of using the native producer directly.

First, E. coli is genetically tractable, grows quickly, and has well-established molecular biology tools. It is much easier to modify promoters, ribosome binding sites, plasmid copy number, codon usage, and pathway architecture in E. coli than in many native producers.

Second, E. coli is a standard chassis for metabolic engineering. It can be used to systematically tune enzyme expression and optimize flux through a pathway.

Third, using E. coli allows researchers to modularize the pathway and test how each genetic part affects production. This makes it a powerful platform for learning, engineering, and scaling bioproduction.

Committed Listener Questions

1. What are the enzymes of the carotenoid pathway?

The carotenoid pathway used in this experiment includes the following enzymes:

A simplified pathway is:

FPP → GGPP → phytoene → lycopene → beta-carotene

where:

crtE: FPP → GGPP
crtB: GGPP → phytoene
crtI: phytoene → lycopene
crtY: lycopene → beta-carotene

2. Which step is rate-determining?

In carotenoid biosynthesis, a common bottleneck is the conversion of phytoene to lycopene, catalyzed by CrtI, because this step requires multiple desaturation reactions.

However, the actual rate-limiting step can depend on context. In engineered E. coli, bottlenecks may also arise from limited precursor supply, plasmid burden, enzyme expression imbalance, oxygen availability, or insufficient GGPP production.

For this lab, I would treat CrtI-mediated phytoene desaturation as a likely pathway bottleneck, while also considering precursor supply through CrtE and central metabolism.

3. Which organism would I choose for production: E. coli or S. cerevisiae?

For this experiment, I would choose E. coli.

Reasons:

1. E. coli grows rapidly.
2. Plasmid-based expression is simple and well characterized.
3. Transformation and selection are straightforward.
4. It is compatible with high-throughput screening.
5. It is easier to tune promoters, RBSs, plasmid copy number, and pathway gene expression.

However, S. cerevisiae could be useful for more complex eukaryotic pathways or products requiring organelle-related metabolism, lipid compartments, or eukaryotic post-translational processing.

For carotenoid production as a teaching and optimization experiment, E. coli is the better chassis.

Expression Construct Design

Chosen gene

For a basic expression construct, I would choose:

crtI

because CrtI is responsible for the conversion of phytoene into lycopene and is likely to strongly influence pigment output.

Proposed construct

Promoter — RBS — crtI coding sequence — Terminator — Origin of replication — Antibiotic resistance marker

Construct parts

Promoters

What is the function of a promoter?

A promoter is a DNA sequence that recruits RNA polymerase and initiates transcription. It determines when, where, and how strongly a gene is transcribed.

In metabolic engineering, promoter strength is one of the most important tuning parameters because too little expression may limit production, while too much expression may burden the cell or create toxic intermediates.

What types of promoters exist?

Common promoter types include:

What promoter would be useful to turn off transcription in response to a metabolite?

A repressible promoter or a metabolite-responsive riboswitch/operator system would be useful. In this design, the metabolite would trigger repression of transcription when it accumulates.

What promoter would be useful to increase transcription in response to a metabolite?

An inducible promoter or metabolite-responsive activator system would be useful. In this case, the metabolite would activate gene expression.

What promoter would I choose for crtI?

I would choose an IPTG-inducible promoter, such as T7-lac or pTac, because it allows controlled expression of crtI.

This is useful because carotenoid pathway enzymes may impose metabolic burden. Inducible expression allows cells to grow before strong pathway expression is activated.

Origin of Replication

What is an origin of replication?

The origin of replication is the DNA sequence that allows a plasmid to replicate inside a host cell. It controls plasmid copy number and compatibility with other plasmids.

Types of origins of replication

What are compatibility groups?

Compatibility groups describe whether two plasmids can be stably maintained in the same cell. Plasmids with the same or very similar origins of replication often belong to the same compatibility group and may be unstable together.

If engineering multiple plasmids, it is important to use different compatible origins.

Best origin for this construct

For crtI, I would choose a medium-copy origin, such as p15A, because it provides a balance between expression strength and metabolic burden.

A very high-copy plasmid might increase crtI expression, but it could also overload the cells, reduce growth, or create pathway imbalance.

Other Important Bioparts

Ribosome Binding Site

The RBS controls translation initiation. A strong RBS can increase enzyme production, while a weaker RBS can reduce burden or prevent accumulation of toxic intermediates.

For carotenoid production, RBS tuning is especially important because pathway balance matters. Overexpressing one enzyme while underexpressing another can create bottlenecks.

Terminator

A terminator stops transcription and prevents readthrough into neighboring genetic parts. A strong terminator improves construct insulation and makes expression more predictable.

Operator

An operator is a DNA sequence bound by a transcriptional regulator. It allows inducible or repressible control of transcription.

For example, lac operators can be used for IPTG-regulated expression.

Aptamers and Riboswitches for Metabolic Tuning

Aptamers are nucleic acid sequences that bind specific ligands. Riboswitches are RNA regulatory elements that change structure when they bind a metabolite, thereby controlling gene expression.

In metabolic engineering, riboswitches can be used to create feedback control.

For example, if lycopene or a pathway intermediate accumulates, a riboswitch could reduce expression of an upstream enzyme to avoid metabolic burden or toxic accumulation. Alternatively, a metabolite-responsive switch could increase expression of a downstream enzyme when precursor levels are high.

This type of dynamic control is useful because the optimal enzyme expression level may change during growth.

Assembly Strategy

To build the carotenoid expression construct, several DNA assembly strategies could be used:

For a modular metabolic pathway, I would choose Golden Gate Assembly because it allows standardized assembly of promoter, RBS, coding sequence, and terminator parts.

Before assembly, I would check the selected gene and vector sequences for internal type IIS restriction sites. If internal sites are present, they may need to be silently removed by codon optimization.

CRISPR-Based Metabolic Engineering

The recitation focused on CRISPR gene regulation, especially CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa).

Unlike gene editing, CRISPRi and CRISPRa use catalytically inactive Cas proteins, such as dCas9, to regulate transcription without cutting DNA.

In metabolic engineering, this is useful because the highest expression of every pathway enzyme is not always the best production strategy. Instead, production often requires balanced expression across pathway steps.

For carotenoid production, CRISPRa or CRISPRi could be used to tune genes such as:

crtE, crtB, crtI, crtY, crtZ, crtW

This would allow systematic exploration of pathway expression levels and could help identify combinations that maximize production of lycopene, beta-carotene, zeaxanthin, or astaxanthin.

Dream Bioproduction Pathway

A pathway I would like to engineer is a microbial system for producing portable biosensor reagents or environmentally useful biomolecules, rather than only pigments.

One possible target would be production of components for low-cost diagnostic or environmental biosensing, such as:

1. DNA-binding proteins,
2. reporter enzymes,
3. fluorescent proteins,
4. Cas proteins,
5. or stabilizing proteins for cell-free diagnostic systems.

This connects directly to my final project, where I am developing a DNAzyme–Cas12a amplified sensor for Pb²⁺ detection in water. In the future, engineered microbes or cell-free bioproduction platforms could be used to produce biosensor components locally and at lower cost.

Connection to My Final Project

My final project is focused on a DNAzyme–Cas12a amplified biosensor for Pb²⁺ detection.

Week 12 connects to my project in several ways:

1. Metabolic engineering logic: The same design-build-test logic used to optimize carotenoid production can be applied to optimize biosensor components.
2. Expression tuning: CRISPRi/CRISPRa shows how biological systems can be tuned rather than simply turned on or off.
3. High-throughput screening: The carotenoid lab compares many culture conditions; my sensor could similarly be optimized across Mg²⁺ concentration, pH, reporter concentration, Cas12a concentration, and DNAzyme/trigger stoichiometry.
4. Bioproduction: In the future, biosensor proteins and reagents could be produced using engineered organisms or cell-free systems.
5. Automation: Combining high-throughput screening with automated liquid handling would accelerate optimization of portable environmental biosensors.

Overall, this week helped me think about biological production as an engineering problem: optimizing pathway components, expression levels, host physiology, and measurement strategies to obtain a desired output.