Simple Cloning and DNA Assembly in Escherichia coli by Prolonged Overlap Extension PCR

2013 ◽  
pp. 183-192 ◽  
Author(s):  
Chun You ◽  
Y.-H. Percival Zhang
Keyword(s):  
Escherichia Coli ◽  
Dna Assembly ◽  
Overlap Extension Pcr ◽  
Overlap Extension

scholarly journals Simple Cloning via Direct Transformation of PCR Product (DNA Multimer) to Escherichia coli and Bacillus subtilis

2011 ◽  
Vol 78 (5) ◽  
pp. 1593-1595 ◽  
Author(s):  
Chun You ◽  
Xiao-Zhou Zhang ◽  
Y.-H. Percival Zhang
Keyword(s):  
Escherichia Coli ◽  
Bacillus Subtilis ◽  
Restriction Enzyme ◽  
Dna Fragments ◽  
Direct Transformation ◽  
Content Type ◽  
Overlap Extension Pcr ◽  
Pcr Product ◽  
Overlap Extension ◽  
General Restriction

ABSTRACTWe developed a general restriction enzyme-free and ligase-free method for subcloning up to three DNA fragments into any location of a plasmid. The DNA multimer generated by prolonged overlap extension PCR was directly transformed inEscherichia coli[e.g., TOP10, DH5α, JM109, and BL21(DE3)] andBacillus subtilisfor obtaining chimeric plasmids.

scholarly journals Efficient site-directed mutagenesis using an overlap extension-PCR method for expressing Mycoplasma hyopneumoniae genes in Escherichia coli

2009 ◽  
Vol 79 (1) ◽  
pp. 101-105 ◽  
Author(s):  
Simone Simionatto ◽  
Silvana B. Marchioro ◽  
Vanessa Galli ◽  
Tessália D. Luerce ◽  
Daiane D. Hartwig ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycoplasma Hyopneumoniae ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
Pcr Method

scholarly journals CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

2019 ◽  
Vol 13 (1) ◽  
Author(s):  
Stefano Vecchione ◽  
Georg Fritz
Keyword(s):  
Escherichia Coli ◽  
Synthetic Biology ◽  
Integrated Circuits ◽  
High Efficiency ◽  
Genetic Circuit ◽  
Dna Assembly ◽  
Chromosomal Integration ◽  
Golden Gate ◽  
Genetic Circuits ◽  
E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

scholarly journals Aptamer-dependent full-length cDNA synthesis by overlap extension PCR

BioTechniques ◽  
2004 ◽  
Vol 37 (1) ◽  
pp. 124-129 ◽  
Author(s):  
Yasumasa Mitani ◽  
Takayuki Nakayama ◽  
Matthias Harbers ◽  
Yoshihide Hayashizaki
Keyword(s):  
Full Length ◽  
Cdna Synthesis ◽  
Full Length Cdna ◽  
Overlap Extension Pcr ◽  
Overlap Extension

A Combination Strategy for Construction of Peptide-��2m-H-2Kb Single Chain with Overlap Extension PCR and One-Step Cloning

2016 ◽  
Vol 26 (12) ◽  
pp. 2184-2191 ◽  
Author(s):  
Tao Xu ◽  
Xiaoe Li ◽  
You Wu ◽  
Khawar Ali Shahzad ◽  
Wei Wang ◽  
...  
Keyword(s):  
Single Chain ◽  
Combination Strategy ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
One Step

Implications of Stisa2 catalytic residue restoration through site directed mutagenesis

2016 ◽  
Vol 42 (2) ◽  
Author(s):  
Hasnain Hussain ◽  
Nikson Fatt Ming Chong
Keyword(s):  
Catalytic Activity ◽  
Catalytic Triad ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Qualitative And Quantitative Analysis ◽  
Conserved Residues ◽  
Overlap Extension Pcr ◽  
Overlap Extension ◽  
Catalytic Network ◽  
And Function

AbstractObjective:Restoration of catalytic activity of Isa2 fromMethods:The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed inResults:Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity.Conclusions:These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

Overlap Extension PCR Cloning

2011 ◽  
Vol 1 ◽  
Author(s):  
Mary Johnson
Keyword(s):  
Pcr Cloning ◽  
Overlap Extension Pcr ◽  
Overlap Extension
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