Plasmid-based cloning and propagation of DNA in E. coli is common practice in laboratories and in the biopharmaceutical industry, however, unwanted expression of the heterologous DNA in E. coli can be toxic preventing the replication or resulting in the accumulation of unwanted mutations in the heterologous DNA. These mutations allow the E. coli to replicate the DNA by eliminating the toxicity, but it will result in an unwanted final protein product. This invention makes it possible to efficiently clone toxic heterologous DNA in E. coli by virtually eliminating its expression.
Gene expression in E. coli is silenced by sandwiching the heterologous DNA between 2 or more lac operators in the cloning plasmid and propagating the DNA in a strain that synthesizes the Lac repressor. Depending on the application, additional transcriptional terminator sequences can be included in the regions flanking the heterologous DNA to further increase its stability.
The use of transcriptional regulatory elements to increase the ability to clone and propagate heterologous DNA in E. coli was developed to address difficulties associated with the cloning and stability of influenza hemagglutinin (HA) and neuraminidase (NA) variants into a common reverse genetics vector. The result was a robust viral reverse genetic vector for viral vaccine and antigen production that used this simple approach which can be readily implemented for stabilizing heterologous DNA in any plasmid that is propagated in E. coli.
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Development Stage: Proof-of-concept; Research and Development Tool
Inventors: Robert Daniels (FDA), Tahir Malik (FDA), and Jan-Willem de Gier (Stockholm University)
Publications: Malik T, Klenow L, Karyolaimos A, Gier JW, Daniels R. Silencing Transcription from an Influenza Reverse Genetics Plasmid in E. coli Enhances Gene Stability. ACS Synth Biol. 2023 Feb 17;12(2):432-445.
Intellectual Property: U.S. provisional application 63/346,568 was filed May 22, 2022
Product Area: Vaccine Development; Protein Expression; Cloning; Plasmid; Expression Vector
FDA Reference No: E-2021-019
FDA Technology Transfer Program