Various genetic switches have been developed to let engineered cells perform designed functions. However, a sustained input is often needed to maintain the on/off state, which is energy-consuming and sensitive to perturbation. Therefore, we developed a set of transcriptional switches for cell states control that were constructed by the inversion effect of site-specific recombinases on terminators. Such a switch could respond to a pulse signal and maintain the new state by itself until the next input. With a bottom-up design principle, we first characterized the terminators and recombinases. Then the mutual interference was studied to select compatible pairs, which were used to achieve one-time and two-time state transitions. Finally, we constructed a biological seven-segment display as a demonstration to prove such switch's immense potential for application.
Recombinases ; metabolism

A new neo/E counterselection technique was set up using Red recombination, which could be used in constructing recombinant plasmid. Firstly, linear targeting cassettes were amplified by PCR; secondly, two steps of homology recombination occurred in vivo: (1) The neo/E counterselection targeting cassette, consisting of a unique endonuclease recognition site and an antibiotic resistance gene, was introduced into the targeted region. (2) The neo/E cassette was replaced in the second round of recombination by another linear targeting cassettes DNA fragment carrying the targeted gene. For selecting a correct recombinant plasmid from the mixture of nonrecombinant and recombinant clones, the unique endonuclease recognition site in the nonrecombinant clones was cut by endonuclease and then transformed into the E. coli competent cells, up to 20% correct recombinants were yielded. A recombinant plasmid of pGL3-Basic PC1900T was successfully constructed in this way. Application of this technique offers a new and highly efficient way for recombinant plasmids construction.
Bacteriophage lambda ; genetics ; DNA, Recombinant ; genetics ; Escherichia coli ; genetics ; Genetic Engineering ; Plasmids ; genetics ; Rec A Recombinases ; genetics ; metabolism ; Recombinant Proteins ; biosynthesis ; genetics

Genetically engineered mouse models are commonly preferred for studying the human disease due to genetic and pathophysiological similarities between mice and humans. In particular, Cre-loxP system is widely used as an integral experimental tool for generating the conditional. This system has enabled researchers to investigate genes of interest in a tissue/cell (spatial control) and/or time (temporal control) specific manner. A various tissue-specific Cre-driver mouse lines have been generated to date, and new Cre lines are still being developed. This review provides a brief overview of Cre-loxP system and a few commonly used promoters for expression of tissue-specific Cre recombinase. Also, we finally introduce some available links to the Web sites that provides detailed information about Cre mouse lines including their characterization.
Animals ; Humans ; Mice* ; Recombinases

OBJECTIVETo analyze sequences of the housekeeping genes including recA, dnaE, and mdh in different serogroups or different biotypes of Vibrio cholerae (VC) strains isolated from China.

METHODS AND RESULTSrecA, dnaE, and mdh genes of Vibrio cholerae were obtained by PCR, sequenced and analyzed. Forty-four variable bases were identified in the 500 bases of recA gene of 18 VC strains, and the mutation of only 3 variable bases could result in changes of 2 amino acids. In the 600 bases of dnaE genes of 18 strains, 32 variable bases were found and only 1 contributed to an amino acid change. In the 367 bases of mdh genes of 18 strains, only 1 variable base was identified whose mutation involved an amino acid convertion. Toxic EI Tor biotype (EVC) strains and toxic O139 serogroup strains were closely related. Non-toxic strains of different serogroups or types were lowly related. Non-toxic and toxic strains of different serogroups or types were lowly related.

CONCLUSIONToxic EVC and toxic O139 serogroup strains isolated from different areas of China may evolve from a common original strain, and toxic O139 VC strains may come from toxic EVC strains.

Bacterial Proteins ; genetics ; Base Sequence ; China ; DNA Polymerase III ; genetics ; Gene Expression Profiling ; Humans ; Molecular Sequence Data ; Phylogeny ; Rec A Recombinases ; genetics ; Sequence Analysis, DNA ; Sequence Homology, Nucleic Acid ; Vibrio cholerae ; classification ; genetics ; isolation & purification

Red/ET recombination, a powerful homologous recombination system based on the Red operon of lambda phage or RecE/ RecT from Rac phage, provides an innovative approach for DNA engineering. Deletion, insertion and mutation can be quickly and precisely performed on the target gene mediated by Red/ET recombination with PCR derived DNA fragments or oligonucleotides. This technical platform has extensive applications in biomedical field including bacterial artificial chromosome modification, gene knock-out construction and genetic modification of E. coli strains as well as some other kinds of microorganisms. Recently, Red/ET recombination was improved in several aspects so that it becomes more powerful and maneuverable. The characteristic and development of Red/ET recombination and its biomedical applications were described in this review.
Bacteriophage lambda ; genetics ; DNA, Recombinant ; genetics ; DNA-Binding Proteins ; genetics ; Escherichia coli ; genetics ; metabolism ; Escherichia coli Proteins ; genetics ; Exodeoxyribonucleases ; genetics ; Genetic Engineering ; methods ; Rec A Recombinases ; genetics ; Recombination, Genetic ; genetics

OBJECTIVETo construct a red fluorescent shuttle vector controlled by recA operon promoter to transform Streptococcus mutans.

METHODSThe promoter of recA was amplified from Streptococcus mutans UA159, and connected to plasmid pDsRed2-N1 to construct pRred with a red fluorescent coding gene, which was then inserted into the shuttle vector pDL276 to construct pLRred.

RESULTSpLRred was successfully constructed, and Escherichia coli transformed with the pLRred plasmid could express reporter gene DsRed.

CONCLUSIONSThe recombination plasmid pLRred can be used in the further research of the expression of cariogenic virulence factor gene by Streptococcus mutans in biofilm.

Escherichia coli ; genetics ; metabolism ; Fluorescent Dyes ; Genes, Essential ; Genes, Reporter ; Genetic Vectors ; Luminescent Proteins ; genetics ; Operon ; Plasmids ; Promoter Regions, Genetic ; Rec A Recombinases ; genetics ; metabolism ; Recombinant Proteins ; genetics ; metabolism ; Streptococcus mutans ; genetics ; Transformation, Bacterial

Animal models, particularly pigs, have come to play an important role in translational biomedical research. There have been many pig models with genetically modifications via somatic cell nuclear transfer (SCNT). However, because most transgenic pigs have been produced by random integration to date, the necessity for more exact gene-mutated models using recombinase based conditional gene expression like mice has been raised. Currently, advanced genome-editing technologies enable us to generate specific gene-deleted and -inserted pig models. In the future, the development of pig models with gene editing technologies could be a valuable resource for biomedical research.
Animals ; Gene Expression ; Gene Transfer Techniques* ; Mice ; Models, Animal ; Recombinases ; Swine

Animals ; Biological Assay ; Nucleic Acid Amplification Techniques ; Phlebovirus/isolation & purification* ; Psychodidae/virology* ; Recombinases/metabolism* ; Reverse Transcription

Objective: To establish an ultra-sensitive, ultra-fast, visible detection method for Vibrio parahaemolyticus (VP) . Methods: We established a new method for detecting the tdh and trh genes of VP using clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 12a (CRISPR/Cas12a) combined with recombinase polymerase amplification and visual detection (CRISPR/Cas12a-VD). Results: CRISPR/Cas12a-VD accurately detected target DNA at concentrations as low as 10 ^-18 M (single molecule detection) within 30 min without cross-reactivity against other bacteria. When detecting pure cultures of VP, the consistency of results reached 100% compared with real-time PCR. The method accurately analysed pure cultures and spiked shrimp samples at concentrations as low as 10 ² CFU/g. Conclusion The novel CRISPR/Cas12a-VD method for detecting VP performed better than traditional detection methods, such as real-time PCR, and has great potential for preventing the spread of pathogens.
CRISPR-Cas Systems ; Nucleic Acid Amplification Techniques/methods* ; Recombinases/genetics* ; Vibrio parahaemolyticus/genetics*

Antimicrobial resistance has become a major public health issue of global concern. Conjugation is an important way for fast spreading drug-resistant plasmids, during which the type Ⅳ pili plays an important role. Type Ⅳ pili can adhere on the surfaces of host cell and other medium, facilitating formation of bacterial biofilms, bacterial aggregations and microcolonies, and is also a critical factor in liquid conjugation. PilV is an adhesin-type protein found on the tip of type Ⅳ pili encoded by plasmid R64, and can recognize the lipopolysaccharid (LPS) molecules that locate on bacterial membrane. The shufflon is a clustered inversion region that diversifies the PilV protein, which consequently affects the recipient recognition and conjugation frequency in liquid mating. The shufflon was firstly discovered on an IncI1 plasmid R64 and has been identified subsequently in plasmids IncI2, IncK and IncZ, as well as the pathogenicity island of Salmonella typhi. The shufflon consists of four segments including A, B, C, and D, and a specific recombination site named sfx. The shufflon is regulated by its downstream-located recombinase-encoding gene rci, and different rearrangements of the shufflon region in different plasmids were observed. Mobile colistin resistance gene mcr-1, which has attracted substantial attentions recently, is mainly located in IncI2 plasmid. The shufflon may be one of the contributors to fast spread of mcr-1. Herein, we reviewed the discovery, structure, function and prevalence of plasmid mediated shufflon, aiming to provide a theoretical basis on transmission mechanism and control strategy of drug-resistant plasmids.
Plasmids/genetics* ; Proteins/genetics* ; Bacteria/genetics* ; Recombinases ; Genes, Bacterial ; Anti-Bacterial Agents