Nacis
Shanghai

Wet Lab
Wet Lab

Wet Lab

Lab Notes

Project: CaroteneCraft: Synthetic Biology for Vision Protection

Period: November 2024 - September 2025

2024.11-2025.8

Theoretical Learning

1. Purpose

The purpose of this extended period was to establish a robust theoretical foundation in core biological and engineering disciplines essential for our iGEM project. Before initiating practical laboratory work, the team dedicated time to a structured curriculum in microbiology, genetic engineering, synthetic biology, analytical instrumentation, and the CRISPR-Cas9 gene-editing system. This ensures that all subsequent experimental design and execution are informed by a deep understanding of underlying principles, promoting efficiency, safety, and scientific rigor.

2. Ways

Structured Coursework: Utilizing online platforms and textbook studies.
Team Workshops: Weekly team-led seminars where members presented on specific topics, followed by group discussions and problem-solving sessions.

Team workshop session with theoretical learning materials

Figure 1. Team workshop session with theoretical learning materials

2025.4-2025.8

Carrot Planting

Recorded by Rachel Zhong: In order to compare the efficiency and yield of plant extraction and microbial synthesis, we planted carrots in early April. During the cultivation period, we carefully tended to them by watering, fertilizing, thinning, and weeding. Unfortunately, the plants turned yellow in July, seemingly due to disease, which ultimately led to an unsuccessful cultivation.

Host Selection

  1. Evaluated potential chassis: Yarrowia lipolytica, Candida tropicalis, Saccharomyces cerevisiae.
  2. Selected S. cerevisiae due to:
    1. Well-characterized genetics
    2. Ease of genetic manipulation
    3. Biosafety Level 1 (RG1) status
    4. Tolerance test in YPD + 0.2 g/L β-carotene showed acceptable growth.

Recorded by Abi: Pour plates, streak for single colonies to cultivate seed culture, then transfer to an Erlenmeyer flask for incubation.

Abi weighing yeast extract for media preparation

Figure 12. Abi weighing yeast extract for media preparation

Abi was weighing the yeast extract:

Strain cultivation and concentration determination:

Cell concentration measurement results

Figure 2. Cell concentration measurement results

Pathway Design & Protein Modeling

  1. Designed de novo β-carotene biosynthetic pathway:
    1. Used endogenous mevalonate pathway for precursor supply
    2. Search the information of carB (phytoene desaturase) from Mucor lusitanicus and carRP (bifunctional phytoene synthase/lycopene cyclase) from Mucor circinelloides
  2. Performed protein structure modeling for key enzymes (CarB, CarRP) using Chai Discovery.
  3. Analyzed lipid droplet (LD) targeting signals (HD1-HD4) and predicted their fusion compatibility with GFP and carotenogenic enzymes.

Gene Synthesis & Primer Design

  1. Retrieved gene sequences from NCBI (AJ238028.1, AJ250827.1)
  2. Codon-optimized carB and carRP for S. cerevisiae
  3. Ordered synthetic genes from commercial vendor
  4. Designed primers for PCR amplification and Gibson assembly
  5. Designed homologous arms for POX1 locus integration

Plasmid Construction & Transformation

Recorded by David:

1. Constructed integration cassettes via fusion PCR:

David performing PCR amplification

Figure 7. David performing PCR amplification

2. Cloned into T-vector and transformed into E. coli JM109 (Researcher: David)

3. Extracted plasmid DNA for subsequent yeast transformation

Here is the manual for the gel extraction kit; you can perform DNA purification by following the steps.

Strain Construction SC-1 & LD Signal Validation

  1. Construction of SC-1 strain — a first-generation β-carotene producer The S. cerevisiae BY4741 strain was electroporated with an integration cassette along with the plasmid pRS426-ΔPOX1. Transformants were selected on minimal medium (MM), and positive clones were identified through colony PCR and sequencing. Successful excision of the URA3 marker was confirmed using FOA plates.
Lab instructor explaining integrated gene construction in *S. cerevisiae*

Figure 14. Lab instructor explaining integrated gene construction in S. cerevisiae

The lab instructor is explaining the principle of constructing integrated genes in Saccharomyces cerevisiae.

  • For the extraction of plasmid pRS426-ΔPOX1, please follow the detailed steps provided in the manual. Important: After adding RNase A, the Buffer P1 must be stored at 4°C.
Plasmid extraction kit components

Figure 15. Plasmid extraction kit components

  1. colony PCR verification

Preparing the gel for nucleic acid electrophoresis

Preparing agarose gel for nucleic acid electrophoresis

Figure 18. Preparing agarose gel for nucleic acid electrophoresis

During the construction of the SC-1 strain, 20 single colonies were selected and subjected to colony PCR.

Colony PCR results from 20 selected colonies

Figure 19. Colony PCR results from 20 selected colonies

Colony PCR

  1. Confirmed HD2 as the most effective LD-targeting signal

Fused HD1-HD4 signals to GFP ,Verified LD localization via Nile Red co-staining

HD2 LD-targeting signal validation with GFP and Nile Red staining

Figure 20. HD2 LD-targeting signal validation with GFP and Nile Red staining

SC-2 Strain Construction (LD-Targeted)

  1. Fused HD2 to C-termini of CarB and CarRP via GGGGS linkers
  2. Constructed integration cassette (Part5)
  3. Integrated into POX1 locus via CRISPR-Cas9
  4. Verified via diagnostic PCR and sequencing
  5. Constructed SC-2 strain — second-generation LD-targeted producer The construction process of SC-2 is similar to that of SC-1.

β-Carotene Quantification (Shake Flask)

  1. Cultured SC-1 and SC-2 in YPD60 medium (3 days, 30°C, 200 rpm)
  2. Extracted β-carotene using acid hydrolysis and acetone extraction
β-carotene extraction procedure workflow

Figure 21. β-carotene extraction procedure workflow

  1. HPLC analysis (C18 column, 450 nm detection)
HPLC analysis setup with C18 column

Figure 22. HPLC analysis setup with C18 column

  1. Results:
    1. β-Carotene concentration of SC-1: 129.1 mg/L
    2. β-Carotene concentration of SC-2: 314.8 mg/L (2.4-fold increase)

2025.7 - 2025.9

5-L Bioreactor Fermentation (SC-2)

  1. Conducted fed-batch fermentation in 5 L bioreactor
  2. Conditions: 30°C, pH 5.5, DO > 20%, 400-800 rpm, 2-4 vvm
  3. Monitored real-time color change using custom hardware
  4. First run: Peak titer = 1.3 g/L at 192 h

Recorded by Damien: Pre-experiment preparation, Fetmentation in 5-L bioreactor

Damien preparing cultivation materials for bioreactor

Figure 23. Damien preparing cultivation materials for bioreactor

Recorded by Peiyang:

Peiyang preparing cultivation materials

Figure 24. Peiyang preparing cultivation materials

Peiyang preparing cultivation materials
Peiyang preparing cultivation materials

We conducted the second scale-up fermentation withe the help of a smart monitoring system

Smart monitoring system for second scale-up fermentation

Figure 25. Smart monitoring system for second scale-up fermentation

2025.9

Product Development

  1. Making lipstick infused with β-Carotene
Final product development - lipstick infused with β-carotene

Figure 26. Final product development - lipstick infused with β-carotene