Expression in Escherichia coli of the native cyt1Aa from Bacillus thuringiensis subsp. israelensis

Abstract

The gene cyt1Aa is one of the genes in the complex determining the mosquito larvicidity of Bacillus thuringiensis subsp. israelensis. Previous cloning in Escherichia coli resulted in a 48-bp addition upstream, encoding a chimera. Here, cyt1Aa was recloned without the artifact, and its toxicity against Aedes aegypti larvae and host E. coli cells was retested.

FIG. 1.

Essential features of the nucleotide sequences of the promoter/operator/polylinker region of pUHE-24S (A), pRM4-C (B) and pRM4-C(Nat) (C). P/O, the promoter region with −35 and −10 elements and operator regions (O₁) with transcription start (arrow); RBS, T5 ribosome binding site of the vector and the Shine-Dalgarno-like sequence preceding the cyt1Aa original translation start codon ATG, shown in uppercase. Asterisks designate the centers of symmetry of lacO₁ operators.

FIG. 2.

Immunoblot of Cyt1Aa in clones pRM4-C (even-numbered lanes) and pRM4-C(Nat) (odd-numbered lanes) either uninduced (lanes 1 and 2) or IPTG induced for 1 h (lanes 3 and 4), 2 h (lanes 5 and 6), and 4 h (lanes 7 and 8).

FIG. 3.

Mass growth (A) and viable counts (B) of E. coli clones, induced (open symbols) with IPTG at time zero and uninduced (closed symbols) harboring either pRM4-C (squares) or pRM4-C(Nat) (circles).

FIG. 4.

Mortality of third-instar A. aegypti larvae upon feeding them with a mixture containing the clone producing Cry4Aa and one of the following clones: pRM4-C(Nat) (A), pRM4-C (B), and pUHE-24S (empty vector) (C) at a ratio of 1:4 (by cell number). Samples were added to 20 early-third-instar A. aegypti larvae in disposable cups with 100 ml sterile tap water, and larval mortality was determined after 24 h at 28°C. Standard error values (bars) were calculated from duplicates for each concentration in a single experiment.