How to determine homologous recombination sites for bacterial fluorescent labeling

1. In the so-called site-specific recombination, the relative position of the DNA segments is shifted, resulting in a different rearrangement of the DNA sequence. Site-specific recombination does not depend on the homology of the DNA sequence (although it can also have very short homology sequences), but rather on the presence of DNA sequences that bind to certain enzymes. These specific enzymes catalyze DNA strand breakage and religation, and they initiate site-specific recombination.

In homologous recombination, the severing of the DNA strand is completely random, revealing sequences that bind to proteins such as Reca, leading to cross-recombination. 2. A typical site-specific recombinant system has the following three elements: a specific set of recognition sites, a simple recognition sequence, or a complex structure responsible for the recognition of different protein factors; Recombinant enzymes (SSRSs) that recognize DNA sequences, mediate cleavage relinking, and achieve chain exchange are either single factors in some systems or synergistic with different protein factors to achieve different recombination results. Site-specific recombination requires no DNA synthesis and no energy gain or loss, and requires a special mechanism that specializes in DNA breakage and reconnection to maintain phosphodiester bond balance.

Depending on the sequence and directionality of the recombination site, there are three possible outcomes of site-specific recombination, namely, integration, excision, and inversion

Tell you explicitly that it's a homologous recombination.

Homologous recombination: Recombination between or within DNA molecules.

The bacteria are cultured with fluorescent proteins for a period of time, and then the fluorescent proteins can be seen inside the bacteria!

This exchange is reversible, and the pre-existing DNA sequence is preserved without loss; There is a very short homologous sequence between the phage and the bacterial DNA, and the recombination exchange must pass through one of the specific nucleotides. These two features are common to site-specific recombination.

Good professional question, I read some information. Thirty SD rats were randomly divided into three groups: blank control group, control group, and methotrexate (MTX) group, with 10 rats in each group.

The rats in the control group and the MTX group were given GFP-labeled E. coli TG1 to trace the displaced bacteria, and the rats in the MTX group were injected subcutaneously with MTX to make a rat chemotherapy model. Fluorescence microscopy and plasmid digestion electrophoresis were used to identify whether the bacteria isolated from the viscera were in the intestine. Results:

GFP markers were isolated from mesenteric lymph nodes, liver, spleen, and kidneys of MTX rats. Conclusion: MTX can induce bacterial translocation, and GFP tracing technology is a simple, reliable and effective method in animal studies of bacterial translocation.

Green fluorescent protein, or GFP for short, was first discovered by Osamu Shimomura et al. in 1962 in a jellyfish with the scientific name Aequorea Victoria. The proteins produced by their genes emit green fluorescence when excited by light in the blue wavelength range. This luminescence process also requires the help of the cold light protein aequorin, and this cold light protein can interact with calcium ions (Ca2+).

The wild-type green fluorescent protein found in aequorea victoria, 395 nm and 475 nm, respectively, are the largest and second largest excitation wavelengths, and its emission wavelength peaks at 509 nm, which is weaker in the visible green range. The green luciferin obtained from sea pansy only has a high excitation peak at 498 nm.

In the field of cell biology and molecular biology, the green luciferin gene is often used as a reporter gene. Some modified forms can be used as biological probes, and the green luciferin gene can also be cloned into vertebrates (e.g., rabbits) and used to replicate a hypothetical experimental method.

The specific operation process.

1.I don't know how many antibiotics you've tested, you can try a few more. There are many kinds of antibiotics that I use regularly, ampr, kanr, cmr, puro, hyg, bsd, specr, zeor, you can try them all.

2.Also, I have an idea, just an idea, you can refer to it. Since positive filtering doesn't work, you can try negative filtering, and there are many negative filtering systems to choose from.

You can take the negative screening marker for recombination, dilute the bacterial solution in a gradient and apply it to a non-resistant plate, find a concentration with the right number of colonies, use blotting or directly pick clones, transfer these clones to the negative screening plate, make a good mark, and pick out those clones that cannot grow on the negative screening plate for identification.