Lab (Week 12) — Bioproduction of Beta-Carotene and Lycopene

Completion status:

• This lab was completed theoretically (no physical or virtual wet lab performed).
• All procedures, expected results, and answers below are based on the provided protocol, scientific literature, and standard bioproduction principles.
• The experiment involves genetically modified E. coli with pAC-LYC (lycopene) and pAC-BETA (beta‑carotene) plasmids.

Overview & Objective

We work with E. coli strains carrying either pAC-LYC (lycopene pathway: CrtE, CrtI, CrtB) or pAC-BETA (adds CrtY, converting lycopene to beta‑carotene). Both plasmids confer chloramphenicol resistance. The goal is to optimise pigment production by varying temperature (30°C vs 37°C), growth media (LB, LB+fructose, 2YT, 2YT+fructose), and measuring cell density (OD600) and pigment absorbance (lycopene at 474 nm, beta‑carotene at 456 nm) after acetone extraction.

Pre‑Lab Reading Summary

• Pathway: FPP (farnesyl diphosphate) → (CrtE) → GGPP → (CrtB) → phytoene → (CrtI) → lycopene → (CrtY) → beta‑carotene.
• OD600 measures light scattering by cells, correlating with cell density. Blank with the same media.
• Safety: Acetone is flammable and volatile; use in fume hood, avoid skin contact.

Protocol Part 1: Overnight Cultures (Theoretical Setup)

We set up 16 unique conditions (2 plasmids × 2 temps × 4 media) with duplicates, plus 2 media‑only controls = 34 cultures total.

Each culture: 3 mL media (with chloramphenicol) + 1 µL starter E. coli (specific plasmid). Incubate 24h in roller drum at assigned temperature.

Expected observation (theoretical): After 24h, cultures with growth show colour: lycopene (red‑pink) for pAC-LYC, beta‑carotene (orange‑yellow) for pAC-BETA. Fructose and richer media (2YT) increase cell density and pigment intensity.

Protocol Part 2: OD600 and Pigment Extraction (Theoretical)

OD600 measurement

• Blank spectrophotometer with the appropriate media.
• Measure 800 µL of each culture in a cuvette.
• Record OD600 values (expected: 0.5–3.0 depending on media and temp).

Pigment extraction (acetone method)

For each sample:

1. Transfer 1 mL culture to microcentrifuge tube, centrifuge 14,000 rpm × 1 min, discard supernatant.
2. Repeat twice more (concentrate pellet from 3 mL total culture).
3. Add 700 µL acetone, pipette up/down to resuspend pellet and extract pigments.
4. Centrifuge again, transfer 600 µL coloured supernatant to a new tube.
5. Add 600 µL water (to reduce acetone corrosion).
6. Transfer 1.2 mL to cuvette, measure absorbance at 474 nm (lycopene) for pAC-LYC samples and 456 nm (beta‑carotene) for pAC-BETA samples.

Expected result: Higher absorbance in richer media (2YT) and at 30°C (better folding of pathway enzymes). Fructose may boost production by providing a carbon source that reduces catabolite repression.

Protocol Part 3: Analysis (Theoretical)

Normalise pigment production per cell:
Specific production = (A_pigment) / (OD600)

Example calculation (simulated data):

Conclusion (theoretical): Highest lycopene production (per cell) occurs in 2YT + fructose at 30°C. Beta‑carotene behaves similarly but with lower absolute absorbance due to extra conversion step (CrtY).

Final Results (Example from literature)

The example figure in the protocol shows pAC-BETA performing better at 37°C (contradicting the above). In reality, optimal temperature depends on the specific plasmid and strain. We would plot bar graphs comparing specific production across conditions.

Post‑Lab Questions (Mandatory for All Students)

1. Which genes induce lycopene and beta‑carotene production?

• Lycopene: crtE, crtB, crtI from Erwinia herbicola (pAC-LYC).
• Beta‑carotene: pAC-BETA adds crtY to the above, converting lycopene to beta‑carotene.

2. Why do plasmids need an antibiotic resistance gene?

To select for bacteria that have taken up the plasmid. Only cells with the resistance gene survive on chloramphenicol‑containing media, ensuring all growing cells carry the pigment pathway.

3. Expected outcomes varying media, fructose, temperature?

• Richer media (2YT) → higher cell density (OD600) and generally higher pigment yield.
• Fructose may increase lycopene production by reducing glucose repression and providing a better carbon source for precursor (FPP) supply.
• Lower temperature (30°C) often improves protein folding and activity of the heterologous enzymes, increasing pigment per cell; 37°C may favour growth but lower specific production.

4. What does OD600 measure and how interpreted?

OD600 measures turbidity caused by light scattering from bacterial cells. It correlates with cell concentration (biomass). In this experiment, we normalise pigment absorbance by OD600 to compare production efficiency independent of cell number.

5. Other experimental setups using acetone to separate cellular matter?

• Chlorophyll extraction from plant tissues or algae.
• Lipid extraction for fatty acid analysis (though hexane/isopropanol is more common).
• Steroid hormone extraction from cell cultures.
• Carotenoid extraction from any biological sample (e.g., tomato, carrot).

6. Why engineer E. coli instead of using natural Erwinia herbicola?

• E. coli is better characterised, grows faster, has simpler genetics, and is safer (BSL‑1). It allows easier metabolic engineering, higher titres, and scalable industrial production. Erwinia may have lower yields or produce unwanted side products.

Post‑Lab Questions (Committed Listeners Only)

Enzymes of the carotene pathway

• CrtE (geranylgeranyl pyrophosphate synthase) – converts FPP to GGPP.
• CrtB (phytoene synthase) – condenses two GGPP to phytoene.
• CrtI (phytoene desaturase) – introduces four double bonds to produce lycopene.
• CrtY (lycopene cyclase) – cyclises lycopene to beta‑carotene.

Rate‑determining step

The CrtB (phytoene synthase) step is often rate‑limiting because condensation of two GGPP molecules is thermodynamically unfavourable and slow. CrtI can also be limiting in some backgrounds.

Choice of organism (E. coli vs S. cerevisiae)

E. coli is faster, cheaper, easier to scale, and does not require eukaryotic post‑translational modifications. However, it lacks internal membrane compartments and may accumulate toxic intermediates. S. cerevisiae has endogenous isoprenoid pathway (ergosterol) and can be engineered for higher flux, plus it is GRAS. For lycopene/beta‑carotene, E. coli is more common for industrial production due to rapid growth and simple fermentation. I would choose E. coli for this lab because the pathway enzymes are bacterial (Erwinia), and we already have the plasmids.

Promoter questions

What is the function of a promoter?
A promoter is a DNA sequence that binds RNA polymerase and initiates transcription of a downstream gene.

Types of promoters:
Constitutive (always on), inducible (regulated by small molecules or physical signals), repressible (off in presence of repressor), tissue‑specific (eukaryotes).

To turn off transcription in response to a metabolite: Use a repressible promoter (e.g., Tet‑OFF, LacI‑regulated). To increase transcription in presence of a metabolite: use an inducible promoter (e.g., Tet‑ON, arabinose‑inducible araBAD).

Promoter choice for a carotenoid enzyme (e.g., crtI):
I would use a strong constitutive promoter (e.g., lacUV5 or T5) for high‑level production because the pathway needs high flux. However, if toxicity occurs, use an inducible promoter (e.g., pBAD with arabinose) to separate growth from production.

Origin of replication questions

What is the origin of replication?
A DNA sequence where replication initiates; determines plasmid copy number and compatibility.

Types of origins:
High copy (e.g., pUC – 500–700 copies), medium copy (pBR322 – 15–20 copies), low copy (pSC101 – 5 copies). Also broad‑host‑range (RK2) and single‑stranded (M13).

Compatibility groups: Plasmids with the same origin cannot coexist in the same cell because they compete for replication machinery.

Best origin for the chosen promoter and gene:
For high lycopene production, use high copy origin (pUC or ColE1 derivative) to increase gene dosage. However, too high copy may cause metabolic burden – so medium copy (pBR322) might be better balanced. pAC plasmids already have a p15A origin (low‑medium copy, compatible with ColE1). I would keep the existing origin.

Other bioparts (RBS, terminators, operators)

• RBS (Shine–Dalgarno) : AGGAG – positioned 5‑10 bp upstream of start codon; strength tuned by sequence.
• Terminator : e.g., T7 terminator or rrnB T1T2 – prevents read‑through transcription.
• Operator : LacO for LacI binding – allows inducible repression.

For the crtI gene, I would use a medium‑strength RBS (e.g., from pET system) and a double terminator.

Aptamers and riboswitches (hot – extra points)

Aptamers are short RNA or DNA sequences that bind specific ligands. Riboswitches are natural mRNA regulatory elements with an aptamer domain that changes secondary structure upon ligand binding, controlling transcription termination or translation initiation. They can be used for metabolic tuning by linking production of pathway enzymes to the concentration of an intermediate, creating feedback control without requiring external inducers.

Joining parts together (restriction sites analysis)

We would use Golden Gate assembly (Type IIs restriction enzymes, e.g., BsaI) or Gibson assembly. In silico, check for unwanted restriction sites in the chosen gene and backbone using Benchling. For example, crtI from Erwinia has no BsaI sites, so we can design overhangs for modular assembly.

Extra hot: dream biosynthetic pathway

I would engineer E. coli to produce artemisinic acid (precursor to antimalarial artemisinin). The pathway from S. cerevisiae (AMR1, ADS, CYP71AV1, CPR) would be codon‑optimised and expressed under inducible promoters. This bio‑product could provide low‑cost, high‑purity artemisinin for malaria treatment, bypassing plant extraction.

For S. cerevisiae integration cassette (extra points)

Chromosome integration site: Use delta sequences (long terminal repeats of Ty retrotransposons) – multiple copies exist, allowing multicopy integration. Or use a safe harbour like HO locus or INT1. Cassette design:
Homology arm (500 bp) – Promoter (e.g., TEF1) – Gene (e.g., crtI) – Terminator (CYC1) – Selectable marker (HIS3) – Homology arm.
Use CRISPR‑Cas9 to target the chosen site.

Bioparts for yeast design

• Promoter: Constitutive (TEF1, GPD) or inducible (GAL1, MET17).
• Kozak sequence: AAAAAATG (eukaryotic translation initiation context).
• Terminator: CYC1, ADH1.
• Marker: URA3, HIS3, LEU2 for auxotrophic complementation.

Integration site choice

Ty delta sites are excellent because they are repeated (≈30 copies) and allow high‑copy integration without antibiotics. Also safe harbour HO (mating‑type switching locus) is transcriptionally silent.

DNA sequence for Twist synthesis (hot! extra points)

For a crtI integration cassette in yeast (simplified example):