A thermally stable temperature-controlled pyrolysis system based on λci857/pl and its construction method and application

Abstract

The invention belongs to the field of bioengineering, and in particular relates to a thermally stable temperature-controlled cracking system based on λcI857 / pL and its construction method and application. The thermostable temperature-controlled lysis system based on λcI857 / pL disclosed by the present invention, that is, the plasmid pKF396ML, has a sequence as shown in SEQ ID NO.1. In the plasmid pKF396ML, the cleavage gene E is cloned between the EcoRI and KpnI restriction sites behind the promoter pL in the plasmid pKF396M-5, and the -35 base of the promoter pL is mutated from T to C obtained. The system can still stably inhibit the expression of gene E at a temperature of 37°C, and can still induce bacterial lysis when the temperature exceeds its maximum repression temperature, which is conducive to the rapid growth of bacteria and the maintenance of important surface antigenic determinants, and can Reduce the heat shock response caused by traditional induction methods (28°C → 42°C).

Description

technical field [0001] The invention belongs to the field of bioengineering, and specifically relates to a thermally stable temperature-controlled cracking system based on λcI857 / pL, and also relates to its construction method and application. Background technique [0002] The slough is a complete bacterial empty shell without cytoplasmic content formed after the Gram-negative bacteria are cleaved by the induced expression product of the bacteriophage PhiX174 cleavage gene E. The cleavage gene E encodes a membrane protein containing 91 amino acids, which can be fused to the inner and outer membranes of Gram-negative bacteria and form a specific transmembrane channel with a diameter of about 40-200 nm on the cell membrane. The cytoplasmic contents of Gram-negative bacteria are discharged from the pores, thereby forming bacterial empty shells without nucleic acids, ribosomes and other components (Witte et al., 1990a; Witte et al., 1990b; Witte et al. ., 1992). [0003] Becau...

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Problems solved by technology

[0006] For this reason, some researchers have mutated and modified the pR promoter through random mutation screening and site-directed mutation verification, by mutating T at position -41 of the λpR promoter operating region to C, or mutating A at position -32 to G , the system can still stably inhibit the expression of gene E at 37°C and 38°C, and the combined mutation of the two can still stably inhibit the expression of gene E at a "high temperature" of 39°C. It can still induce bacterial lysis normally when the maximum repression temperature is exceeded (Jechlinger et al., 1999; Jechlinger et al., 2005), but the research is currently limited to the single expression vector of the pR promoter, and there is no information about the pL promoter. Mutation modification and related reports on the construction of temperature-controlled lysis system based on it, and the temperature-controlled system based on λcI857 / pL is also very common in practical applications

In addition, the size of the plasmids involved in the aforementioned studies is large, which is not conducive to the efficient preparation of sloughs; the temperature interval setting for mutation screening of the promoter pR is also "too large", and the set screening intervals overlap, respectively. 37 ℃ ~ 42 ℃ and 38 ℃ ~ 42 ℃, in theory, it will lead to heavy screening tasks, and further improvement is needed

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