New vectors for direct cloning of PCR products

Cha, Jooyeun; Bishai, William R.; Chandrasegaran, Srinivasan

doi:10.1016/0378-1119(94)90148-1

Cited by 8 publications

(7 citation statements)

References 2 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…As a result, a 40-amino-acidresidue sequence, TTMTTMTTEQLGMNNSVNDKLDVLLIGAGFTG LYQLYHLR, was obtained by Edman degradation using an automated protein sequencer (model 477; Perkin-Elmer). Two primers with the following sequence were designed: 5Ј-ACIACIATGACIACNATGAC-3Ј and 5Ј-ARRTGRTAIA RYTGRTA-3Ј, where I ϭ inosine; N ϭ T, C, A, or G; R ϭ A or G; and Y ϭ C or T. The amplified 116-bp product was cloned directly in the PCR product cloning vector pXcmkn12 (14) and transformed in E. coli XL1-Blue, and the resulting plasmid was designated pCMP10 (Table 1; Fig. 2).…”

Section: Methodsmentioning

confidence: 99%

Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli -Expressed Cyclopentanone 1,2-Monooxygenase

Iwaki

Hasegawa

Wang

et al. 2002

Appl Environ Microbiol

125

View full text Add to dashboard Cite

54 -dependent regulatory elements in front of the divergently transcribed cpnB and cpnC genes supports the notion that cpnR is a regulatory gene of the NtrC type. Knowledge of the nucleotide sequence of the cpn genes was used to construct isopropyl-␤-thio-D-galactoside-inducible clones of Escherichia coli cells that overproduce the five enzymes of the cpn pathway. The substrate specificities of CpnA and CpnB were studied in particular to evaluate the potential of these enzymes and establish the latter recombinant strain as a bioreagent for Baeyer-Villiger oxidations. Although frequently nonenantioselective, cyclopentanone 1,2-monooxygenase was found to exhibit a broader substrate range than the related cyclohexanone 1,2-monooxygenase from Acinetobacter sp. strain NCIMB 9871. However, in a few cases opposite enantioselectivity was observed between the two biocatalysts.Pseudomonas sp. strain NCIMB 9872, which is capable of growth on 0.1% cyclopentanol as sole carbon source, was isolated from a freshwater stream in Illinois by P. J. Chapman some three decades ago. Trudgill and coworkers (27) used this strain as a prototype organism to establish the biochemical pathway of cyclopentanol metabolism shown in Fig. 1. For reason of utility as a Baeyer-Villiger (BV) biocatalyst and its mechanistic aspects (2,8,13,58,71), the second biochemical step catalyzed by cyclopentanone 1,2-monooxygenase (CPMO) has been most extensively studied in this organism. The conversion of cyclopentanone to 5-valerolactone is NADPH and flavin adenine dinucleotide (FAD) dependent, and CPMO has been purified to near homogeneity. It is a tetramer of subunit molecular weight 50,000 to 54,000 (8,66). Assay of the FAD/ protein ratio gave values of 2 to 4 molecules of FAD bound to each tetrameric enzyme molecule (28,66). An N-terminal 29-amino-acid sequence of CPMO has been determined (8).A chemical BV oxidation is the transformation of ketones into esters or of cyclic ketones into lactones by peracids, such as 3-chloroperbenzoic acid. This century-old reaction (for reviews, see references 54 and 63) continues to attract interest not only in broadening the spectrum of applications (ranging from the synthesis of steroids, antibiotics, and pheromones to the synthesis of monomers for polymerization, etc.) but also in developing oxidants that are more chemoselective and efficient and thus result in more product than waste (20).The biological BV reactions catalyzed by BV monooxygenases (BVMOs) offer the prospect of eco-efficient chemical transformations in whole cells or improved expression systems via cloning. Moreover, BVMOs can provide products in optically active form which are difficult to obtain by other strategies (for reviews, see references 55, 61, 71, and 74). Notable ste-* Corresponding author. Mailing address:

show abstract

Section: Methodsmentioning

confidence: 99%

Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli -Expressed Cyclopentanone 1,2-Monooxygenase

Iwaki

Hasegawa

Wang

et al. 2002

Appl Environ Microbiol

125

View full text Add to dashboard Cite

show abstract

“…PCR fragments were amplified from the C. acetobutylicum adc promoter using primers ADC55 and ADC3 (323 bp) and from the C. beijerinckii ptb promoter using primers PTB5 and PTB3 (252 bp) and cloned into Sma I‐cut pUC19 and Xcm I‐cut pXcmKm12 (Cha et al ., 1993), respectively, to generate pADCwt and pPTBwt. A B. subtilis abrB promoter fragment (331 bp) to be used as a control was excised from pJM5134 (Perego et al ., 1988) with Dra I– Bam HI and subcloned into pUC19 cut with Sma I– Bam HI to generate pABRB1.…”

Section: Methodsmentioning

confidence: 99%

Spo0A directly controls the switch from acid to solvent production in solvent‐forming clostridia

Ravagnani

Jennert

Steiner

et al. 2000

Molecular Microbiology

137

128

View full text Add to dashboard Cite

SummaryThe spo0A genes of Clostridium beijerinckii NCIMB 8052 and Clostridium cellulolyticum ATCC 35319 were isolated and characterized. The C-terminal DNA-binding domains of the predicted products of spo0A from these two organisms, as well as 16 other taxonomically diverse species of Bacillus and Clostridium, show extensive amino acid sequence conservation (56% identity, 65% similarity over 104 residues). A 12-amino-acid motif (SRVERAIRHAIE) that forms the putative DNA recognition helix is particularly highly conserved, suggesting a common DNA target.

show abstract

“…Such mutations can be identified by high-throughput genome sequencing and confirmed by PCR and dideoxy chain-termination sequencing. The pKSKI series could be made into T-vectors by simple digestion (34)(35)(36), so donor plasmids could be constructed by direct T-A cloning. A resistance gene cassette and I-SceI recognition sites could be integrated into the gene locus of the recipient strain by -Red PCR targeting.…”

Section: Discussionmentioning

confidence: 99%

High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

Yang

Sun

Huang

et al. 2014

Appl Environ Microbiol

View full text Add to dashboard Cite

show abstract

New vectors for direct cloning of PCR products

Cited by 8 publications

References 2 publications

Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli -Expressed Cyclopentanone 1,2-Monooxygenase

Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli -Expressed Cyclopentanone 1,2-Monooxygenase

Spo0A directly controls the switch from acid to solvent production in solvent‐forming clostridia

High-Efficiency Scarless Genetic Modification in Escherichia coli by Using Lambda Red Recombination and I-SceI Cleavage

Contact Info

Product

Resources

About