DNA cloning

Pwo DNA polymerase is an enzyme that is widely used in PCR due to its thermal stability and proofreading activity (3′-5′ exonuclease activity) with high accuracy. Optimizing the expression and purification of the protein is crucial to reducing the cost of production for this important enzyme. In this study, the higher expression level of the gene encoding Pwo DNA polymerase in bacteria strain E. coli BL21(DE3) was achieved by optimizing codon usage.

11p xuanphongdacy06 18-09-2024 2 1   Download

Bài viết Chứng minh BAC clone nhằm dòng hóa vùng chứa gen quy định mùi thơm trên giống lúa Oryza sativa L. trình bày việc thanh lọc thư viện BAC với chỉ thị liên kết với mùi thơm; Xác định những dòng BAC dự tuyển ở locus mùi thơm; Xây dựng BAC contig (các chuỗi DNA nhân bản nằm liền kề) trên vùng gen mùi thơm; Xác định BAC DNA chứa alen mùi thơm;...

6p visybill 19-07-2023 6 2   Download

In this study, the DNA fragment encoding for the C-terminus domain of the AopB from Aeromonas hydrophila AH-1 was cloned into pET-M expression vector and expressed in Escherichia coli BL21 (DE3) host cells. The recombinant AopB-C-terminus domain was successfully purified using immobilized nickel affinity chromatography as a soluble form. Crosslinking analysis among AopB-C-terminus molecules in solution showed that this domain existed as a mixture of tetramer, trimer, dimer, and monomer forms.

6p guitaracoustic02 08-12-2021 18 3   Download

In this study, we evaluated the reliability of genomic DNAs and cloned recombinant plasmids as standard controls for absolute real-time PCR assay. The associated standard curves were constructed and used for the quantification of Lactobacillus acidophilus probiotics.

4p mudbound 10-12-2021 32 2   Download

Bài giảng Sinh học phân tử: Chương 7 Kỹ thuật tạo dòng, cung cấp cho người học những kiến thức như: Các công cụ sử dụng trong kỹ thuật tạo dòng; Vai trò sinh học của RE; Tạo dòng phân tử (molecular cloning); Sự khác nhau của các vector tạo dòng; Plasmid là các vector tạo dòng;...Mời các bạn cùng tham khảo!

60p caphesuadathemmatong 25-11-2021 29 2   Download

Bài giảng Sinh học phân tử: Chương 7 trình bày về kỹ thuật tạo dòng. Chương này trình bày những nội dung chính sau: Các công cụ sử dụng trong kỹ thuật tạo dòng, Restriction Enzyme (RE), vai trò sinh học của RE, restriction endonuclease, tạo dòng phân tử (molecular cloning), DNA tái tổ hợp, công cụ tạo dòng,... Mời các bạn tham khảo.

34p hihihaha2 03-12-2016 95 12   Download

Bài giảng "Simple cloning" cung cấp cho người học các kiến thức: Vector pUC, vai trò của các gene, cấu trúc của các gen, cắt vector và DNA, nối bằng ligase được, chuyển tất cả cấu trúc vào E.coli, chọn dòng mang DNA tái tổ hợp, vector nhân tạo đầu tiên, tế bào chủ, tính chất của tế bào chủ, nhân dòng sản phẩm PCR,... Mời các bạn cùng tham khảo.

55p doinhugiobay_12 15-01-2016 55 5   Download

Bài giảng "Công nghệ di truyền - Chương 3: Simple cloning" cung cấp cho người học các kiến thức: Vector pUC, vai trò của các gene, cắt vector và DNA, nối bằng ligase được, chuyển tất cả cấu trúc vào E.coli, Operon lacZ và IPTG, chọn dòng mang DNA tái tổ hợp,... Mời các bạn cùng tham khảo nội dung chi tiết.

55p doinhugiobay_10 12-01-2016 111 19   Download

An optogenetic upgrade for the tet - OFF system fused the inhibitor to the blue light-responsive B-LID degron and optimized the performance of this construct with regard to the number of Med25VBD repeats. This approach resulted in an optogenetic upgrade of the popular Tet-OFF (TetR-VP64, tetO7-PhCMVmin)  system  that  allows tunable, blue light-inducible gene expression in HEK-293T  cells.

5p thuyancn 01-06-2015 30 5   Download

Telomerase is a specialized reverse transcriptase responsible for synthesizing telomeric DNA at the ends of chromosomes. Six subunits composing the telomerase complex have been cloned: hTR (human telomerase RNA), TEP1 (telomerase-associated protein 1), hTERT (human telomerase reverse transcriptase), hsp90 (heat shock protein 90), p23, and dyskerin. In this study, we investigated the role of each the telomerase subunit on the activity of telomerase.

9p research12 01-06-2013 28 3   Download

We report the cloning and functional charaterization of the full-length cDNA and gene encoding a Toxoplasma gondii DNA repair enzyme designated TgDRE. The gene is composed of three exons separated by two introns of 780 and 630 bp, and encodes a protein with a predicted molecular mass of 49.6 kDa. The native TgDRE protein, with a molecular mass of 60 kDa, is only detected in the virulent tachyzoite stage of T. gondii. However, the transcript is present in both asexual parasite stages, virulent tachyzoite and avirulent encysted bradyzoite. ...

9p research12 01-06-2013 33 5   Download

Primary structural information including the complete nucleotide sequence of the ®rst insect aldehyde oxidase (AO) was obtained from the common house mosquitoCulex quinquefasciatus(Say) through cloning and sequencing of both genomic DNA and cDNA. The deduced amino-acid sequence encodes a 150-kDa protein of 1266 amino-acid residues, which is consistent with the expected monomeric subunit size of AO.

12p research12 01-06-2013 48 5   Download

We have cloned the gene coding for theBacillus subtilis glycine oxidase (GO), a new ¯avoprotein that oxidizes gly-cine and sarcosine to the corresponding a-keto acid, ammonia and hydrogen peroxide. By inserting the DNA encoding for GO into the multiple cloning site of the expressionvectorpT7.7weproducedarecombinantplasmid (pT7-GO).ThepT7-GOencodes a fullyactive fusionprotein with six additional residues at the N-terminus of GO (MARIRA).

8p research12 29-04-2013 38 3   Download

The primary purpose of the present study was to investi-gate whether DNA replication at meiotic prophase also requires replication factors, especially proliferating cell nuclear antigen (PCNA). We cloned PCNA cDNAs (CoPCNA) from a cDNA library made from basidia of the basidiomycete,Coprinus cinereus. Interestingly, although CoPCNAis a single-copy gene in the genome, two di€er-entPCNAcDNA species were isolated using degenerate primers and a meiotic cDNA library, and were designated asCoPCNA-aandCoPCNA-b. ...

11p research12 29-04-2013 42 4   Download

Thea3b1 integrin is an adhesion receptor for extracellular matrix proteins including isoforms of laminin, and the changes of its expression level in various cancer cells are thought to cause their malignant phenotypes. We have cloned an approximately 4 kb DNA fragment of the 5¢-flanking region of the murinea3 integrin gene and analyzed its promoter activity.

9p research12 23-04-2013 36 3   Download

A nitroalkane-oxidizing enzyme gene (naoA) was cloned from a genomic DNA library ofStreptomyces ansochromo-genes7100. The deduced protein (NaoA) of this gene con-tains 363 amino acids and has high similarity to several nitroalkane-oxidizing enzymes from various micro-organ-isms.ThenaoAgenewas subcloned intoanexpressionvector pET23b and overexpressed inEscherichia coliBL21(DE3). The protein was then purified, and its characteristics were studied. Experimental results showed that NaoA can con-vert 1-nitropropane, 2-nitropropane and nitroethane into the corresponding carbonyl compounds....

6p tumor12 22-04-2013 44 2   Download

Human cytochrome P450 17a-hydroxylase (CYP17) cata-lyses not only the 17a-hydroxlation of pregnenolone and progesterone and the C17,20-side chain cleavage (lyase) of 17a-hydroxypregnenolone, necessary for the biosynthesis of C21-glucocorticoids and C19-androgens, but also catalyses the 16a-hydroxylation of progesterone. In efforts to under-stand the complex enzymology of CYP17, structure/func-tion relationships have been reported previously after expressingrecombinant DNAs, encodingCYP17 from various species, in nonsteroidogenic mammalian or yeast cells. ...

9p tumor12 22-04-2013 42 2   Download

Wehave identifiedanovelmammaliangene, termednicolin1 gene (NICN1), that is present in human, dog and mouse, whereas it is absent from the available genome sequences of nonmammalian organisms. TheNICN1gene consists of six exons and spans about 6 kb of genomic DNA. It encodes a 213 amino acid protein that does not belong to any known protein family. Experiments using green fluorescent protein (GFP)-taggednicolin1 fusionproteins indicate that nicolin1 is a nuclear protein.

6p tumor12 22-04-2013 51 2   Download

We have isolated and studied the cloned sigma factors SASIG1-3 from mustard (Sinapis alba). In functional ana-lyses using both promoter and factor mutants,the three recombinant proteins all had similar basic properties but also revealed differences in promoter preference and requirements for single nucleotide positions. Directedmuta-genesis of SASIG1 identified critical residues within the conserved regions 2.4 and 4.2 necessary for binding of the )10 and)35 promoter elements,respectively.

13p tumor12 20-04-2013 33 2   Download

We have previously cloned, expressedand characterized two variants of the major allergen Lep d 2 from culturedLepi-doglyphus destructor mites. These variants, Lep d 2.0101 and Lep d 2.0201, differ at 13 amino acid positions. In this study we investigated Lep d 2 sequence diversity between wild and cultured mites. PCR, Southern blot and DNA sequence analysis revealed the presence of two different Lep d 2 genes, one with and one without an intron.

8p tumor12 20-04-2013 47 3   Download