![]() Recent work by our laboratory has significantly expanded the capacity of GGA by using ligase fidelity data to select fusion sites, a process termed data-optimized assembly design (DAD), with the successful assembly of 35 fragments into a 5 kb cassette in a single reaction using this strategy. This strategy has worked well for small DNA targets but generally limits users to <10 fragments. (9) To avoid erroneous assembly products caused by ligation errors, GGA is typically limited to sets of overhang sequences that contain multiple mismatches between all non-cognate pairs or pre-vetted sets of fusion sites. T4 DNA ligase is the most frequently utilized DNA ligase in GGA, as it joins assembly fragments more efficiently and with less bias than other commercially available DNA ligases however, T4 DNA ligase is prone to ligation of some mismatched sequences, which can result in constructs with mis-ordered, duplicated, or missing assembly pieces. (16) Another limitation of GGA is that the method relies on the accurate and efficient ligation of assembly pieces by a DNA ligase to avoid improperly ordered fragments and low assembly yield. Recent work using methylation to protect recognition sequences from digestion offers an additional solution to this problem. Typically, this limitation is overcome by introducing silent mutations to remove internal sites within coding regions mutagenesis outside coding regions can require guesswork and careful control to remove internal sites without significant perturbation to the system. While GGA permits scarless assembly even within coding regions, the desired assembly sequence cannot contain the recognition sequence of the type IIS restriction enzyme used in the assembly, as these sites would be cleaved in the final assembly. Recent work suggests that GGA can routinely accommodate many additional fragments per reaction, potentially reducing the number of, or even entirely avoiding, multiple assembly rounds. (6,7) Thus, hierarchical assembly schemes involving multiple rounds of molecular cloning, construct purification, and sequence verification are employed for the in vitro assembly of large constructs. (2−5) Nevertheless, in vitro DNA assembly methods, such as Golden Gate assembly (GGA), remain popular to generate constructs due to their ease of use despite typical standards being limited to 5–10 DNA fragments per reaction. (1) For example, in vivo recombination methods permit assembly of >200 kb from as many as 50 fragments in a single transfection. Typically, these constructs are much too long to be directly synthesized and are consequently assembled from multiple shorter DNA fragments. Large DNA constructs are widely used in synthetic biology to develop genetically engineered organisms for therapeutic uses and chemical production. The assembly protocols and design principles described here can be applied to rapidly engineer a wide variety of large and complex assembly targets. We applied these insights to genome construction, successfully assembling the 40 kb T7 bacteriophage genome from up to 52 parts and recovering infectious phage particles after cellular transformation. While GGA is routinely used to generate constructs from 5 to 10 DNA parts in one step, we found that optimization permitted the assembly of >50 DNA fragments in a single round. To address this problem, we sought to test whether Golden Gate assembly (GGA), an in vitro DNA assembly methodology, can be utilized to construct a large DNA target from many tractable pieces in a single reaction. However, the manufacture of these constructs is laborious, often involving multiple hierarchical rounds of preparation. ![]() Large DNA constructs (>10 kb) are invaluable tools for genetic engineering and the development of therapeutics. ![]()
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