No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology

Abstract

The Registry of Standard Biological Parts imposes sequence constraints to enable DNA assembly using restriction enzymes. Alnahhas et al. (Journal of Biological Engineering 2014, 8:28) recently argued that these constraints should be revised because they impose an unnecessary burden on contributors that use homology-based assembly. To add to this debate, we tested four different homology-based methods, and found that students using these methods on their first attempt have a high probability of success. Because of their ease of use and high success rates, we believe that homology-based assembly is a best practice of Synthetic Biology, and recommend that the Registry implement the changes proposed by Alnahhas et al. to better support their use.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-015-0006-z) contains supplementary material, which is available to authorized users.

Improvement of the reverse tetracycline transactivator by single amino acid substitutions that reduce leaky target gene expression to undetectable levels

“…Overlap extension PCR was used to create all synthetic DNA constructs 28 . DNA constructs containing the yeGFP, GEV and rtTA parts were sequentially integrated into the genome at the ADE2 , GAL4 and ADE4 loci respectively, of S. cerevisiae strain BY4742 (MAT α , his3Δ1, leu2Δ0, lysΔ0, ura3Δ0) (EUROSCARf).…”

Engineered gene networks enable non‐genetic drug resistance and enhanced cellular robustness

“…Strains were created by integrating synthetic cassettes into the genome through a combination of polymerase chain reaction (PCR), DNA splicing by overlap extension PCR, and transformation‐associated recombination (TAR) [37, 38]. Briefly, DNA sequences were cloned by PCR to add overhangs of 20–50 base pairs, homologous to either an adjacent DNA sequence in the synthetic cassette or the region of the yeast genome into which the entire cassette would be inserted.…”

Erratum to: No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology

“…After the publication of this work [ 1 ], we noticed that an incorrect version of Fig. 1 was published.…”