36 results about "Double crossover" patented technology

A method for the positive selection of allelic exchange mutants is provided for C1 metabolizing bacteria. The chromosomal integration vectors, based on the pGP704 suicide vector, comprise at least one genetic selectable marker and the sacB gene, encoding levansucrase. A one- and two-step selection strategy is provided for the facile identification of double-crossover mutations in C1 metabolizing bacteria. This methodology enables production of “markerless” transformants, such that multiple rounds of mutation can be performed. Optimized conditions for conjugal transfer, homologous recombination, transformant purification, and screening are also presented.

Robust nucleic acid arrays and lattices are assembled based on double crossover cohesion of polygonal units whose edges are composed of nucleic acid multi-crossover domains.

The invention relates to an urban railway double-crossover middle turning device and a method for eliminating lateral bending and upwarping of a beam body. A butt joint part between a first rotary beam and a second rotary beam is of a lug boss lap-joint structure and is located on a base through a rotary table, the lower part of a non-butt-joint part of each of the first rotary beam and the second rotary beam is provided with a trolley moving mechanism, the trolley moving mechanisms are located on bases, a drive guide slot is arranged at the bottom of each of the first rotary beam and the second rotary beam, one ends of two drive arms are respectively located in the drive guide slots, the other ends of the two drive arms are respectively connected with a power output end of a respective reduction gear, the power input ends of the reduction gears are respectively connected with a power output end of a double-end motor reduction gear through a transmission shaft and a transmission shaft support frame, two locking frames are respectively located on the bases for supporting the non-butt-joint parts of the first rotary beam and the second rotary beam, and two locking mechanisms are respectively located at the back parts of the non-butt-joint parts of the first rotary beam and the second rotary beam and are respectively matched with the locking frames in a locking or opening manner.

The invention discloses a method of performing traceless knockout on a chromogene or inserting an exogenous gene in a chromosome of Acidithiobacillus caldus. The method comprises the following steps: transferring a homologous recombinant plasmid containing plentiful restriction enzyme digestion sites of endonuclease I-SceI into the Acidithiobacillus caldus in a conjugation transfer manner, simultaneously, directionally inserting the homologous recombinant plasmid into the chromosome of Acidithiobacillus caldus by use of a homologous recombinant system of a cell itself, transferring a screened single crossover to an I-SceI expression plasmid by an electroporation method, performing secondary homologous recombination on a single recon chromosome under a stress that the I-SceI enzyme cuts the chromosome, and optimizing and screening a double crossover bacterial strain to realize knockout and insertion of the gene. The invention also constructs a suicide type plasmid vector pSDUDI for homologous recombination and a plasmid pSDU1-I-SceI for expressing the I-SceI enzyme effectively, and provides convenience for the traceless knockout and the integration of the chromogene in the Acidithiobacillus caldus. The method provided by the invention provides a new way for intensive study on and modification of Acidithiobacillus caldus.

The invention belongs to the technical field of medicines, and relates to an engineering bacterium producing gentamicin A and an application thereof. The engineering bacterium is micromonospora purpurea (Micromonospora purpurea) KD1989 with a geneD1 inactivating function, which is registered and collected in China General Microbiological Culture Collection Center (CGMCC) with the collection No. CGMCC No. 8598. The genD1 function in gentamicin biosynthetic gene cluster is inactivated through a gene knockout method, and according to the gene knockout method, shuttle plasmids (such as pGD14 plasmid) are built and knocked out, the pGD14 plasmid is transformed into a gentamicin producing strain, double crossover engineering bacterium micromonospora purpurea GKD1989(delta genK+delta genD1) is screened, the engineering bacterium is fermented, and gentamicin A is determined by detection of metabolites. The engineering bacterium producing gentamicin A produces the single component gentamicin A, which has not been reported in China and foreign countries, so that the industrial production of gentamicin A can be realized; and the production process is simple and easily controlled, the extraction is convenient, the cost is low, and the engineering bacterium meets clean production levels, has stable product quality, and is the first innovative product in the world.

The invention provides a gene of coded 6-phosphogluconate dehydrogenase and application thereof. The nucleotide sequence is shown in SEQ ID NO:1. The 6-phosphogluconate dehydrogenase gene pgdhD2 is derived from Methylobacterium sp. MB200 and has the L-serine inhibiting effect. The Methylobacterium sp. MB200 is used as an original strain, pgdh gene deletion mutation DMB is built through a homologous double-crossover method, an L-serine high-yield mutant strain with the L-serine producing capacity greatly higher than that of original strain Methylobacterium sp. MB200 is obtained, and the tolerated D-serine concentration of the L-serine high-yield mutant strain is greatly higher than that of the original strain. The pgdh gene deletion mutation stain has the good application prospect in production of L-serine through a fermentation method.