High molecular weight DNA assembly <i>in vivo</i> for synthetic biology applications

Juhas, Mario; Ajioka, James W.

doi:10.3109/07388551.2016.1141394

Cited by 26 publications

(15 citation statements)

References 101 publications

(202 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The Gram‐negative bacterium Escherichia coli and the Gram‐positive bacterium Bacillus subtilis are among the key cellular chassis suitable for a plethora of synthetic biology devices and biotechnology applications (Harwood and Cranenburgh, ; Ajikumar et al ., ; Commichau et al ., ; Juhas and Ajioka, ). Consequently, both E. coli and B. subtilis have been successfully used for the production of a number of the industrially important products, such as biofuels, biosensors, pharmaceuticals and natural dyes (Ajikumar et al ., ; Yim et al ., ; Park et al ., ; Zhou et al ., ; Hao et al ., ; Manabe et al ., ; McKenney et al ., ).…”

Section: Introductionmentioning

confidence: 99%

“…Consequently, both E. coli and B. subtilis have been successfully used for the production of a number of the industrially important products, such as biofuels, biosensors, pharmaceuticals and natural dyes (Ajikumar et al ., ; Yim et al ., ; Park et al ., ; Zhou et al ., ; Hao et al ., ; Manabe et al ., ; McKenney et al ., ). Furthermore, both E. coli and B. subtilis are considered to be promising chassis for engineering of the minimal and tailor‐made cell factories (Commichau et al ., ; Juhas et al ., ; Juhas, ; Juhas and Ajioka, ). The majority of the good DNA editing techniques have been developed in E. coli ; however, B. subtilis has many advantages.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

T7 RNA polymerase‐driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis

Juhas

Ajioka

2017

Microbial Biotechnology

Self Cite

View full text Add to dashboard Cite

SummaryThe majority of the good DNA editing techniques have been developed in Escherichia coli; however, Bacillus subtilis is better host for a plethora of synthetic biology and biotechnology applications. Reliable and efficient systems for the transfer of synthetic DNA between E. coli and B. subtilis are therefore of the highest importance. Using synthetic biology approaches, such as streamlined lambda Red recombineering and Gibson Isothermal Assembly, we integrated genetic circuits pT7L123, Repr‐ts‐1 and pLT7pol encoding the lysis genes of bacteriophages MS2, ΦX174 and lambda, the thermosensitive repressor and the T7 RNA polymerase into the E. coli chromosome. In this system, T7 RNA polymerase regulated by the thermosensitive repressor drives the expression of the phage lysis genes. We showed that T7 RNA polymerase significantly increases efficiency of cell lysis and transfer of the plasmid and bacterial artificial chromosome‐encoded DNA from the lysed E. coli into B. subtilis. The T7 RNA polymerase‐driven inducible cell lysis system is suitable for the efficient cell lysis and transfer of the DNA engineered in E. coli to other naturally competent hosts, such as B. subtilis.

show abstract

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

T7 RNA polymerase‐driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis

Juhas

Ajioka

2017

Microbial Biotechnology

Self Cite

View full text Add to dashboard Cite

show abstract

“…Advances in biology and biotechnology have led to development of a variety of methods for assembly of large DNA constructs ( 1 , 2 ). Existing methods can be largely divided into two groups.…”

Section: Introductionmentioning

confidence: 99%

MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

O’Callaghan

2018

Nucleic Acids Research

View full text Add to dashboard Cite

Efficient DNA assembly is of great value in biological research and biotechnology. Type IIS restriction enzyme-based assembly systems allow assembly of multiple DNA fragments in a one-pot reaction. However, large DNA fragments can only be assembled by alternating use of two or more type IIS restriction enzymes in a multi-step approach. Here, we present MetClo, a DNA assembly method that uses only a single type IIS restriction enzyme for hierarchical DNA assembly. The method is based on in vivo methylation-mediated on/off switching of type IIS restriction enzyme recognition sites that overlap with site-specific methylase recognition sequences. We have developed practical MetClo systems for the type IIS enzymes BsaI, BpiI and LguI, and demonstrated hierarchical assembly of large DNA fragments up to 218 kb. The MetClo approach substantially reduces the need to remove internal restriction sites from components to be assembled. The use of a single type IIS enzyme throughout the different stages of DNA assembly allows novel and powerful design schemes for rapid large-scale hierarchical DNA assembly. The BsaI-based MetClo system is backward-compatible with component libraries of most of the existing type IIS restriction enzyme-based assembly systems, and has potential to become a standard for modular DNA assembly.

show abstract

“…DNA sequence assembly, which refers to the precise aligning and merging multiple fragments of DNA, in an end-to-end fashion, into large synthetic circuits and pathways, plays a pivotal role in protein structure-function, metabolic engineering, and synthetic biology [ 1 – 5 ]. The increasingly high demand for assembling large DNA into functional devices requires the methods that allow scarless, sequence independent, multi-fragment assembly of large constructs at high efficiency and high fidelity [ 6 , 7 ].…”

Section: Introductionmentioning

confidence: 99%

AFEAP cloning: a precise and efficient method for large DNA sequence assembly

et al. 2017

View full text Add to dashboard Cite

BackgroundRecent development of DNA assembly technologies has spurred myriad advances in synthetic biology, but new tools are always required for complicated scenarios. Here, we have developed an alternative DNA assembly method named AFEAP cloning (Assembly of Fragment Ends After PCR), which allows scarless, modular, and reliable construction of biological pathways and circuits from basic genetic parts.MethodsThe AFEAP method requires two-round of PCRs followed by ligation of the sticky ends of DNA fragments. The first PCR yields linear DNA fragments and is followed by a second asymmetric (one primer) PCR and subsequent annealing that inserts overlapping overhangs at both sides of each DNA fragment. The overlapping overhangs of the neighboring DNA fragments annealed and the nick was sealed by T4 DNA ligase, followed by bacterial transformation to yield the desired plasmids.ResultsWe characterized the capability and limitations of new developed AFEAP cloning and demonstrated its application to assemble DNA with varying scenarios. Under the optimized conditions, AFEAP cloning allows assembly of an 8 kb plasmid from 1-13 fragments with high accuracy (between 80 and 100%), and 8.0, 11.6, 19.6, 28, and 35.6 kb plasmids from five fragments at 91.67, 91.67, 88.33, 86.33, and 81.67% fidelity, respectively. AFEAP cloning also is capable to construct bacterial artificial chromosome (BAC, 200 kb) with a fidelity of 46.7%.ConclusionsAFEAP cloning provides a powerful, efficient, seamless, and sequence-independent DNA assembly tool for multiple fragments up to 13 and large DNA up to 200 kb that expands synthetic biologist’s toolbox.Electronic supplementary materialThe online version of this article (doi: 10.1186/s12896-017-0394-x) contains supplementary material, which is available to authorized users.

show abstract

High molecular weight DNA assembly in vivo for synthetic biology applications

Cited by 26 publications

References 101 publications

T7 RNA polymerase‐driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis

T7 RNA polymerase‐driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis

MetClo: methylase-assisted hierarchical DNA assembly using a single type IIS restriction enzyme

AFEAP cloning: a precise and efficient method for large DNA sequence assembly

Contact Info

Product

Resources

About