Experimental Techniques

The commonly used chassis cells in synthetic biology include Escherichia coli, yeast, Bacillus subtilis, etc. Cell transformation, positive strain screening, and genetic stability verification are the core steps in strain construction, with the focus being on improving transformation efficiency, reducing false positives, and ensuring genetic stability of the strain.

1. Fine-tuning techniques for competent cell transformation

• Cell state control: Homemade competent cells should strictly control the OD600 at 0.4–0.6. At this point, the cells are in the logarithmic growth phase, and the cell wall permeability is optimal, resulting in the highest transformation efficiency; an OD value that is too high leads to cell aging and insufficient cell viability, both of which will significantly reduce the transformation rate.

• Transformation system standardization: The amount of plasmid added should be controlled at 1–5 μL (concentration 50–100 ng/μL). Excessive plasmid addition can cause cell poisoning and an increase in false positives; the heat shock temperature and time must be matched with the strain: for Escherichia coli, 42°C heat shock for 90 seconds, for yeast, 40°C heat shock for 30 seconds. Do not exceed the time or temperature limits.

• Key points for recovery culture: Immediately add LB medium without antibiotics after heat shock, incubate on a shaker at 37°C for 60 minutes, at a low speed (150 rpm), to ensure cell repair and expression of resistance genes, and direct plating will lead to a decrease in the survival rate of positive strains.

2. Techniques for positive strain screening and verification

• Optimization of plate screening: The antibiotic concentration in the resistance plate is strictly standardized. For Escherichia coli, ampicillin 50 μg/mL, kanamycin 100 μg/mL, and chloramphenicol 25 μg/mL. Excessive concentration inhibits the growth of weakly expressed strains, while too low a concentration leads to contamination by miscellaneous bacteria and false positives.

• Dual verification mechanism: For the initial screening of single colonies, a colony PCR verification should be performed first to quickly screen for positive clones; then, extract the plasmid for enzyme digestion verification and sequencing verification to eliminate issues such as fragment insertion misalignment, base mutations, and fragment deletion. Synthetic biology engineered strain must undergo sequencing verification to be preserved.

• Preservation of strain: Verified strains are preserved using 20% glycerol at low temperatures, for short-term storage (1–3 months) at -20°C, and for long-term storage (more than 1 year) at -80°C; do not perform multiple passages to avoid plasmid loss and element mutations, ensuring experimental reproducibility.