Stably self-luminous bacteria plasmid and preparation method thereof

Abstract

The invention relates to a stably self-luminous bacteria plasmid and a preparation method thereof. The stably self-luminous bacteria plasmid comprises a luxABCDE self-luminous gene, a promoter capable of starting the luxABCDE self-luminous gene, a ColE1 replicon, an ampicillin resistant gene, a kanamycin resistant gene, stable plasmid correlated genes and conjugation effect correlated genes. The stably self-luminous bacteria plasmid of the invention can easily enter into bacteria through conjugational transfer to express a luminous gene protein and stably stay in bacteria in quantity. Besides, the stably self-luminous bacteria plasmid can be luminous continuously and efficiently without addition of extra luminous substrates or screening of antibiotic, so as to facilitate direct observation of bacteria dynamic change in an animal in vivo. The preparation method of the invention comprises the following steps of: a. acquiring a plasmid capable of starting a luminous gene; b. inserting a plasmid containing the ColE1 replicon and the resistant gene into a plasmid pSE34; c. combining the plasmids from step a. and step b. to obtain the plasmid of the invention.

Description

technical field [0001] The invention discloses a luminescent plasmid that can be used to calibrate bacteria and a preparation method thereof, especially technically providing a gram-negative bacterium that can be stable in bacterial reproduction from generation to generation and exists in large quantities in bacteria, and a preparation method thereof. Background technique [0002] Generally, if you want to know the distribution and behavior of bacteria in animals, you can only analyze animal organ specimens after the sacrifice of experimental animals, and you cannot directly observe them. [0003] Therefore, some people have developed a method of expressing luminescent genes in bacteria to observe the distribution and behavior of bacteria in animals. Generally speaking, there are three main ways to express luminescent genes in bacteria: [0004] 1. Directly express the luminescent gene in the plasmid, but due to the instability of the plasmid itself, most bacteria are easy t...

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Problems solved by technology

[0004] 1. Directly express the luminescent gene in the plasmid, but due to the instability of the plasmid itself, most bacteria are easy to lose the implanted plasmid, and cannot be stable and exist in a large number of bacteria, but in the reproduction of bacteria , it is difficult for the plasmid to exist stably in each daughter bacterium, and it can only be maintained for a few generations, so the strength of the overall bacterium will soon weaken

Antibiotic selection is used to force the bacteria to retain the plasmid, otherwise they cannot survive, but this method cannot be used in live animals, because if the animals are fed antibiotics, the variability caused by the antibiotics to the test animals may complicate the experimental results

[0005] 2. The luminescent gene is inserted into the bacterial chromosome opportunistically through the transposon, but it is not sure whether the inserted DNA is where the important gene is located; and because the probability of transposition is not high, more screening is required difficulty of

Moreover, this method is only for strains that have been transposed. If it is applied to other strains, the positions of the transposons will be different. This will add more variables, so that it is impossible to observe and compare multiple strains.

[0006] 3. Insert the luminescent gene into the bacterial chromosome by double group exchange. Although it can be accurately inserted into a specific gene, the probability of its occurrence is not high, and because the luminescent gene (luxABCDE-kan) itself is very long (at least 7kb), it is even lower. The probability of recombination and exchange makes it difficult to complete the cloning and delivery of macromolecular DNA in bacteria

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