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Synthetic biology focuses on quantitative expression, controllable expression, and efficient synthesis of products. The temperature, rotation speed, induction timing, and concentration of inducers during strain cultivation directly affect the yield of target proteins and metabolites, and are key steps in engineering phenotypic characterization.
1. Standardized techniques for shake flask cultivation
• Inoculation ratio standardization: Activate the bacterial strain at a 1%–2% inoculation ratio and inoculate the liquid medium. After overnight activation, transfer the culture to a larger volume at a ratio of 1:100 to ensure synchronous cell growth and avoid uneven expression due to differences in bacterial growth.
• Precise control of cultivation parameters: The basic cultivation temperature is 37°C (for bacteria) and 30°C (for yeast). The rotation speed is 220 rpm, ensuring adequate dissolved oxygen. Insufficient dissolved oxygen can lead to anaerobic metabolism of the cells, slow growth, and a decrease in the synthesis of target products.
• Control of growth curve: Real-time monitoring of OD600 values. The induction of expression should be strictly selected at the mid-logarithmic growth phase (OD600 = 0.6–0.8), where cell vitality is the strongest, the transcription and translation system is active, and the efficiency of induction expression is the highest. Inducing too early results in low cell concentration, and inducing too late leads to aging of the cells and a decrease in expression levels.
2. Optimization techniques for controllable induction expression
• Appropriate concentration of inducers: The conventional final concentration of IPTG induction is 0.1–1.0 mM. Weak promoters should use high concentrations (0.8–1.0 mM), and strong promoters should use low concentrations (0.1–0.3 mM). Excessive induction can lead to cytotoxicity and cell lysis.
• Quality control at low temperatures: When expressing toxic proteins or proteins that form inclusion bodies, induce the cells at a temperature of 16–25°C and incubate at a low speed for 12–16 hours. This significantly increases the proportion of soluble protein expression and reduces the formation of inclusion bodies.
• Blank control setting: Each expression experiment must include an uninduced control group and a vector control group to exclude background expression of the cells and interference from impurities in the culture medium, ensuring the specificity of the expression product and the validity of the experimental data.
