Experimental Techniques

Carrier construction and component assembly are the most fundamental experimental steps in synthetic biology. The precise assembly of standardized components such as promoters, RBS, terminators, and coding sequences directly determines the expression effect of subsequent engineered strains. The key is to avoid issues such as base mutations, frameshift mutations, self-ligation of the vector, and misalignment of components.

1. Standardized techniques for primer design (specific to synthetic biology)

Unlike ordinary PCR primer design, the cloning of synthetic biology components must take into account compatibility, ligation efficiency, absence of redundant sequences, and expression adaptability.

• Core parameter control: The length of the primers is controlled within 25–35 bp, the difference in annealing temperature is ≤ 1°C, ensuring uniform PCR amplification; the GC content is 40%–60%, avoiding high GC hairpin structures at the upstream and downstream of the functional elements, and preventing amplification truncation. The overlapping region of the splicing primers strictly retains 15–20 bp homologous arms, achieving the optimal homologous recombination efficiency.

• Precise distinction of component boundaries: When designing, strictly define the boundaries of the promoter, RBS, CDS, and terminator, and prohibit the introduction of restriction enzyme sites or homologous sequences within the functional elements to avoid disrupting the function of the elements; common standardized restriction enzyme sites (EcoRⅠ, BamHⅠ, XhoⅠ) are uniformly placed in the flanking regions of the elements, with 4–6 bp of protection bases reserved to ensure sufficient ligation.

• Key points to avoid pitfalls: Absolutely avoid primer dimerization (ΔG < -5 kcal/mol), otherwise it will significantly reduce the amplification efficiency of the target fragment; before amplifying the exogenous gene, the template plasmid sequence must be sequenced for verification to prevent template mutations from causing the failure of element function.

2. Practical techniques for homologous recombination assembly (seamless cloning)

Seamless cloning is the mainstream method for the tandem assembly of multiple components in synthetic biology. Compared to traditional restriction enzyme ligation, it has no base redundancy and can achieve one-time assembly of multiple fragments. The key to practical operation lies in the fragment concentration ratio and system optimization.

• Quantitative ratio principle: For single-fragment assembly, the molar ratio of the vector to the inserted fragment is 1:2; for 2–3 fragment series assembly, the molar ratio of the vector: fragment 1: fragment 2 = 1:2:3, and the molar ratio of multiple fragments increases successively, ensuring that small fragments preferentially bind to the vector to improve the assembly success rate.

• System pre-treatment: The target fragment after PCR amplification must be recovered by gel and purified to remove residual primers, dNTPs, and polymerase to avoid interfering with the activity of the recombinase; the vector plasmid must be completely linearized, and non-complete restriction enzyme-cut vectors must be avoided to prevent false positive self-ligation of the vector.

• Precise control of reaction conditions: The recombinant system is prepared at low temperature on ice to avoid denaturation at room temperature; the reaction time is 30 minutes at 25°C for common fragments, extended to 45 minutes for high GC and long fragments (> 2000 bp); the reaction is immediately terminated on ice after completion to prevent non-specific ligation.