Recombinational Cloning
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Abstract The study of protein en masse , or functional proteomics, depends on the availability of full‐length cDNAs in appropriate expression‐ready plasmid vectors for protein expression and functional analysis. Recombinational cloning is a universal cloning technique based on site‐specific recombination that is independent of the insert DNA sequence to be cloned, which differentiates this method from the classical restriction enzyme‐based cloning methods. Recombinational cloning enables rapid and efficient parallel transfer of DNA inserts into multiple expression systems. This unit summarizes strategies for generating expression‐ready clones using two of the most popular recombinational cloning technologies, now commercially available from Invitrogen (Gateway) and BD Clontech (Creator).Keywords:
Cloning (programming)
Insert (composites)
Plasmid preparation
Agarose gel electrophoresis
Strain (injury)
T-DNA Binary system
Transduction (biophysics)
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DNA nanostructures have been shown viable for the creation of complex logic-enabled sensing motifs. To date, most of these types of devices have been limited to the interaction with strictly DNA-type inputs. Restriction endonuclease represents a class of enzyme with endogenous specificity to DNA, and we hypothesize that these can be integrated with a DNA structure for use as inputs to trigger structural transformation and structural rearrangement. In this work, we reconfigured a three-arm DNA switch, which utilizes a cyclic Förster resonance energy transfer interaction between three dyes to produce complex output for the detection of three separate input regions to respond to restriction endonucleases, and investigated the efficacy of the enzyme targets. We demonstrate the ability to use three enzymes in one switch with no nonspecific interaction between cleavage sites. Further, we show that the enzymatic digestion can be harnessed to expose an active toehold into the DNA structure, allowing for single-pot addition of a small oligo in solution.
Exogenous DNA
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Abstract IncX4 plasmids are one of the most epidemiologically successful vehicles for mcr-1 spread. Here we found that the IncX4 plasmids carried two different replication proteins encoded by genes pir-1 and pir-2 , respectively, but mcr-1 was only carried by IncX4 plasmid encoding pir-1 . The copy number of pir-2 encoding plasmids (3.15±0.9 copies) are higher than that of pir-1 encoding plasmids (0.85±0.5 copies). When mcr-1 was cloned into IncX4 plasmid encoding pir-2 , the higher copy number of these plasmids resulted in increased expression of mcr-1 and a greater fitness burden on their host cells. However, these plasmids exhibited a lower rate of invasion into the bacterial population compared to mcr-1 positive plasmids encoding the pir-1 gene. These findings collectively explain the absence of mcr-1 in all IncX4 plasmids encoding pir-2 . Our results further confirmed that low-copy numbers are important for the spread of mcr-1 plasmid from the perspective of natural evolution.
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IncX4 plasmids are one of the most epidemiologically successful vehicles for mcr-1 spread. Here we found that the IncX4 plasmids carried two different replication proteins encoded by genes pir-1 and pir-2, respectively, but mcr-1 was only carried by IncX4 plasmid encoding pir-1. The copy number of pir-2 encoding plasmids (3.15±0.9 copies) are higher than that of pir-1 encoding plasmids (0.85±0.5 copies). When mcr-1 was cloned into IncX4 plasmid encoding pir-2, the higher copy number of these plasmids resulted in increased expression of mcr-1 and a greater fitness burden on their host cells. However, these plasmids exhibited a lower rate of invasion into the bacterial population compared to mcr-1 positive plasmids encoding the pir-1 gene. These findings collectively explain the absence of mcr-1 in all IncX4 plasmids encoding pir-2. Our results further confirmed that low-copy numbers are important for the spread of mcr-1 plasmid from the perspective of natural evolution.
Replicon
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Insert (composites)
Package insert
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Restriction map
Restriction fragment
Plasmid preparation
Restriction digest
Restriction site
Alkaline lysis
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This paper make the insert design as masterstroke,not only describes the condition of the injection-mould insert design but also the insert's advantages and the disadvantages;the application of the insert designing and how to design the insert's fixation and anti-rotation.
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Package insert
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We have developed a novel protocol for directional cloning of PCR products into any vector. The target sequence is amplified in two parallel asymmetric PCRs using specially but simply designed primers. Two single-stranded products are produced and they are annealed to form a double-stranded DNA fragment bearing overhangs at both ends that correspond to the restriction overhangs of certain restriction enzymes. The fragment can then be cloned into a certain vector previously treated with the corresponding enzymes without restriction of the inserted fragment. Compared with previously published protocols, the procedure described in this paper is highly efficient and it is independent of the restriction sites of the insert and is therefore applicable to molecular biology and biotechnology studies.
Insert (composites)
Cloning (programming)
Restriction site
Fragment (logic)
Cloning vector
Restriction digest
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본 연구에서는 새롭게 개발된 "강도 향상 인써트"에 대해 조인트 시편을 제작하여 Pull-out 및 전단시험을 수행하여 보통 인써트의 조인트 강도와 비교하고 그 파손 특성을 조사하였다. 이를 통해 새로운 인써트에 대한 성능을 확인하였다. 시험 결과, 새로운 "강도 향상 인써트"는 기존의 보통 인써트에 비해 Pull-out 강도는 2.1배, 전단강도는 2.04배 향상되는 것을 확인하였다. 따라서 향후 높은 인써트 강도를 요구하는 구조에 유용하게 사용될 수 있을 것이다. In this study, joint strength and failure characteristics were experimentally examined with pull-out and shear specimens in which new designed "high strength insert" was applied. The performance of the new insert was compared with typical insert design. The experimental results showed that the "high strength insert" had the joint strengths of 2.1 times in the pull-out specimens and 2.04 times in the shear specimen compared with typical insert joints. Therefore, the new developed "high strength insert" will be usefully used in the aerospace structure.
Insert (composites)
Shear Strength
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