Bacillus expression system

Inventors

TABOR, Jeffrey J. • CASTILLO-HAIR, Sebastian M.

Assignees

William Marsh Rice University

Abstract

An inducible promoter expression system based on the T7 RNA Polymerase (T7 RNAP), lactose repressor (Lad), and a chimeric T7lac promoter (PT7lac), which can be integrated as a single copy into the B. subtilis genome. In the absence of IPTG, Lad strongly represses T7RNAP and PT7lac, and expression of an exemplary ORF—here superfolder green fluorescent protein (sfGFP) reporter protein—is undetectable by flow cytometry. Addition of IPTG de-represses PT7lac, and simultaneously induces expression of T7RNAP, resulting in very high sfGFP levels.

Core Innovation

An inducible promoter expression system based on the T7 RNA Polymerase (T7 RNAP), lactose repressor (LacI), and a chimeric T7lac promoter (PT7lac), which can be integrated as a single copy into the B. subtilis genome. In the absence of IPTG, LacI strongly represses T7RNAP and PT7lac, and expression of an exemplary ORF—here superfolder green fluorescent protein (sfGFP) reporter protein—is undetectable by flow cytometry. Addition of IPTG de-represses PT7lac, and simultaneously induces expression of T7RNAP, resulting in very high sfGFP levels.

The system combines the viral T7 RNAP, which produces high amounts of recombinant protein in the active state, with the LacI bacterial transcription factor that inhibits production of both the protein and the viral polymerase, resulting in nearly undetectable output in the inactive state. The LacI-T7 inducible system comprises the following elements: Transcriptional repressor LacI, expressed constitutively; the T7 phage RNA polymerase (T7 RNAP), expressed from the B. subtilis promoter Phy-spank, which is normally repressed by LacI; and the hybrid PT7lac promoter, normally repressed by LacI as well, under whose control the recombinant protein of interest or ORF is placed.

The background problem being solved is that typical inducible promoters in B. subtilis have dynamic ranges of at most a few hundred and reported T7-based systems exhibit high leakiness, limiting fold-activation and protein yield. The invention addresses the need for a very tightly regulated, high dynamic range, integratable inducible expression system in Bacillus and reports systems achieving greater than 10,000-fold or even 20,000-fold activation in response to a chemical inducer.

Claims Coverage

Three inventive features are identified from two independent claims.

The claims cover an expression system in Bacillus having high sequence identity to SEQ ID NO 7 with ≥95% identity (excluding an added ORF), an IPTG-inducible expression performance of at least 5,000-fold, and a DNA molecule comprising SEQ ID NO 7 plus an added ORF.

Stated Advantages

Documented Applications