39 research outputs found
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T7 RNA polymerase-driven inducible cell lysis for DNA transfer from Escherichia coli to Bacillus subtilis
The 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.This work was supported by the UK Engineering and Physical Sciences Research Council (EPSRC), OpenPlant Fund and SynBio Fund
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Combining Genes from Multiple Phages for Improved Cell Lysis and DNA Transfer from to
The ability to efficiently and reliably transfer genetic circuits between the key synthetic biology chassis, such as and , constitutes one of the major hurdles of the rational genome engineering. Using lambda Red recombineering we integrated the thermosensitive lambda repressor and the lysis genes of several bacteriophages into the chromosome. The lysis of the engineered autolytic cells is inducible by a simple temperature shift. We improved the lysis efficiency by introducing different combinations of lysis genes from bacteriophages lambda, ΦX174 and MS2 under the control of the thermosensitive lambda repressor into the chromosome. We tested the engineered autolytic cells by transferring plasmid and bacterial artificial chromosome (BAC)-borne genetic circuits from to . Our engineered system combines benefits of the two main synthetic biology chassis, and B. subtilis, and allows reliable and efficient transfer of DNA edited in into .This work was supported by grants from the UK Engineering and Physical Sciences Research Council (JWA), OpenPlant Fund (MJ) and SynBio Fund (MJ)
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New tools for self-organized pattern formation
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Reverse transcription-polymerase chain reaction construction of plasmid-based, full-length cDNA libraries from Leishmania infantum for in vitro expression screening
Role of monomorphic ChrIa in fixation of global population structure of T. Gondii.
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New tools for self-organised pattern formation
Position-dependent gene expression is a critical aspect of the development and behaviour of multicellular organisms. It requires a complex series of interactions to occur between different cell types in addition to intracellular signalling cascades. We used Escherichia coli to study the properties of an artificial signalling system at the interface between two expanding cell populations. We genetically engineered one population to produce a diffusible acyl-homoserine lactone (AHL) signal, and another population to respond to it. Our experiments demonstrate how such a signal can be used to reproducibly generate simple visible patterns with high accuracy in swimming agar. The producing and responding cassettes of two such signalling systems can be linked to produce a symmetric interface for bidirectional communication that can be used to visualise molecular logic. Intracellular feedback between these two cassettes would then create a framework for self-organised patterning of higher complexity. Adapting the experiments of Basu et al. (Basu et al., 2005) using cell motility, rather than a differential response to AHL concentrations as a way to define zones of response, we noted how the interaction of sender and receiver cell populations on a swimming plate could lead to complex pattern formation. Equipping highly motile strains such as E. coli MC1000 with AHL-mediated auto-inducing systems based on Vibrio fischeri luxI/luxR and Pseudomonas aeruginosa lasI/lasR cassettes would allow the amplification of a response to an AHL signal and its propagation. We designed and synthesised codon-optimised auto-inducing luxI/R and lasI/R cassettes as optimal gene expression is crucial for the generation of robust patterns. We still have to complete and test the entire genetic circuitry, although by modelling the system we were able to demonstrate its feasibility. © 2007 The Institution of Engineering and Technology
Molecular cloning and characterization of mitogen-activated protein kinase 2 in Toxoplasma gondii
Characterization of the pufferfish (Fugu) genome as a compact model vertebrate genome
Nature3666452265-268NATU