Mathematical model of a serine integrase-controlled toggle switch with a single input

Dual-state genetic switches that can change their state in response to input signals can be used in synthetic biology to encode memory and control gene expression. A transcriptional toggle switch (TTS), with two mutually repressing transcription regulators, was previously used for switching between two expression states. In other studies, serine integrases have been used to control DNA inversion switches that can alternate between two different states. Both of these switches use two different inputs to switch ON or OFF. Here, we use mathematical modelling to design a robust one-input binary switch, which combines a TTS with a DNA inversion switch. This combined circuit switches between the two states every time it receives a pulse of a single-input signal. The robustness of the switch is based on the bistability of its TTS, while integrase recombination allows single-input control. Unidirectional integrase-RDF-mediated recombination is provided by a recently developed integrase-RDF fusion protein. We show that the switch is stable against parameter variations and molecular noise, making it a promising candidate for further use as a basic element of binary counting devices

Isolation and Characterisation of E. coli xerC, a Gene Required for cer Site-Specific Recombination

The formation of plasmid multimers by homologous recombination can greatly reduce the heritable stability of the natural multicopy plasmid ColE1. ColE1 carries a 250 bp site (cer) which, when present in direct repeat (as in a ColE1 dimer) efficiently resolves plasmid multimers to momomers. cer-mediated monomerisation is strongly correlated with an increase in plasmid stability. Current evidence suggests that ColE1 segregates randomly at cell division, and that cer-dependent monomerisation ensures stability by increasing the number of independently segregating plasmid copies (Sherratt et a 7, 1984; Summers and Sherratt, 1984)

Rapid metabolic pathway assembly and modification using serine integrase site-specific recombination

Synthetic biology requires effective methods to assemble DNA parts into devices and to modify these devices once made. Here we demonstrate a convenient rapid procedure for DNA fragment assembly using site-specific recombination by ϕC31 integrase. Using six orthogonal attP/attB recombination site pairs with different overlap sequences, we can assemble up to five DNA fragments in a defined order and insert them into a plasmid vector in a single recombination reaction. ϕC31 integrase-mediated assembly is highly efficient, allowing production of large libraries suitable for combinatorial gene assembly strategies. The resultant assemblies contain arrays of DNA cassettes separated by recombination sites, which can be used to manipulate the assembly by further recombination. We illustrate the utility of these procedures to (i) assemble functional metabolic pathways containing three, four or five genes; (ii) optimize productivity of two model metabolic pathways by combinatorial assembly with randomization of gene order or ribosome binding site strength; and (iii) modify an assembled metabolic pathway by gene replacement or addition

Residential development in London in the nineteenth century: A comparative examination of its nature and progress within the present boroughs of Camden and Lambeth

Abstract not provide

A mini-ISY100 transposon delivery system effective in γ proteobacteria

Transposons are invaluable biological tools for the genetic manipulation of microorganisms. ISY100 from Synechocystis sp. PCC6803 is a member of the Tc1/mariner/IS630 superfamily, and is characterized by high transposition efficiency and a strong preference for TA target sequences. In this paper, we describe the design and application of a mini-ISY100 suicide vector for the in vivo creation of stable random transposon insertion libraries. The system was successfully applied in seven species belonging to four different orders of γ proteobacteria. In all cases, delivery using conjugation consistently showed the highest transposition efficiency compared to chemical transformation or electroporation. We determined the frequency of transposon insertions in all the species and proved the utility of the system by identifying genes involved in colony coloration in Shewanella oneidensis. The ease and the efficiency of the protocol developed here allow the creation of complete knock-out libraries in an extensive range of host microorganisms in less than a week with no requirement for preparatory modification

Multipart DNA Assembly Using Site-Specific Recombinases from the Large Serine Integrase Family.

Assembling multiple DNA fragments into functional plasmids is an important and often rate-limiting step in engineering new functions in living systems. Bacteriophage integrases are enzymes that carry out efficient recombination reactions between short, defined DNA sequences known as att sites. These DNA splicing reactions can be used to assemble large numbers of DNA fragments into a functional circular plasmid in a method termed serine integrase recombinational assembly (SIRA). The resulting DNA assemblies can easily be modified by further recombination reactions catalyzed by the same integrase in the presence of its recombination directionality factor (RDF). Here we present a set of protocols for the overexpression and purification of bacteriophage ϕC31 and Bxb1 integrase and RDF proteins, their use in DNA assembly reactions, and subsequent modification of the resulting DNA assemblies

A single-input binary counting module based on serine integrase site-specific recombination

A device that counts and records the number of events experienced by an individual cell could have many uses in experimental biology and biotechnology. Here, we report a DNA-based ‘latch’ that switches between two states upon each exposure to a repeated stimulus. The key component of the latch is a DNA segment whose orientation is inverted by the actions of ϕC31 integrase and its recombination directionality factor (RDF). Integrase expression is regulated by an external input, while RDF expression is controlled by the state of the latch, such that the orientation of the invertible segment switches efficiently each time the device receives an input pulse. Recombination occurs over a time scale of minutes after initiation of integrase expression. The latch requires a delay circuit, implemented with a transcriptional repressor expressed in only one state, to ensure that each input pulse results in only one inversion of the DNA segment. Development and optimization of the latch in living cells was driven by mathematical modelling of the recombination reactions and gene expression regulated by the switch. We discuss how N latches built with orthogonal site-specific recombination systems could be chained together to form a binary ripple counter that could count to 2N − 1

The mechanism of ϕC31 integrase directionality : experimental analysis and computational modelling

Serine integrases, DNA site-specific recombinases used by bacteriophages for integration and excision of their DNA to and from their host genomes, are increasingly being used as tools for programmed rearrangements of DNA molecules for biotechnology and synthetic biology. A useful feature of serine integrases is the simple regulation and unidirectionality of their reactions. Recombination between the phage attP and host attB sites is promoted by the serine integrase alone, giving recombinant attL and attR sites, whereas the 'reverse' reaction (between attL and attR) requires an additional protein, the recombination directionality factor (RDF). Here, we present new experimental data on the kinetics and regulation of recombination reactions mediated by ϕC31 integrase and its RDF, and use these data as the basis for a mathematical model of the reactions. The model accounts for the unidirectionality of the attP × attB and attL × attR reactions by hypothesizing the formation of structurally distinct, kinetically stable integrase-DNA product complexes, dependent on the presence or absence of RDF. The model accounts for all the available experimental data, and predicts how mutations of the proteins or alterations of reaction conditions might increase the conversion efficiency of recombination

mwr Xer site-specific recombination is hypersensitive to DNA supercoiling

The multiresistance plasmid pJHCMW1, first identified in a Klebsiella pneumoniae strain isolated from a neonate with meningitis, includes a Xer recombination site, mwr, with unique characteristics. Efficiency of resolution of mwr-containing plasmid dimers is strongly dependent on the osmotic pressure of the growth medium. An increase in supercoiling density of plasmid DNA was observed as the osmotic pressure of the growth culture decreased. Reporter plasmids containing directly repeated mwr, or the related cer sites were used to test if DNA topological changes were correlated with significant changes in efficiency of Xer recombination. Quantification of Holliday junctions showed that while recombination at cer was efficient at all levels of negative supercoiling, recombination at mwr became markedly less efficient as the level of supercoiling was reduced. These results support a model in which modifications at the level of supercoiling density caused by changes in the osmotic pressure of the culture medium affects resolution of mwr-containing plasmid dimers, a property that separates mwr from other Xer recombination target sites

Gated rotation mechanism of site-specific recombination by ϕC31 integrase

Integrases, such as that of the Streptomyces temperate bacteriophage ϕC31, promote site-specific recombination between DNA sequences in the bacteriophage and bacterial genomes to integrate or excise the phage DNA. ϕC31 integrase belongs to the serine recombinase family, a large group of structurally related enzymes with diverse biological functions. It has been proposed that serine integrases use a “subunit rotation” mechanism to exchange DNA strands after double-strand DNA cleavage at the two recombining att sites, and that many rounds of subunit rotation can occur before the strands are religated. We have analyzed the mechanism of ϕC31 integrase-mediated recombination in a topologically constrained experimental system using hybrid “phes” recombination sites, each of which comprises a ϕC31 att site positioned adjacent to a regulatory sequence recognized by Tn3 resolvase. The topologies of reaction products from circular substrates containing two phes sites support a right-handed subunit rotation mechanism for catalysis of both integrative and excisive recombination. Strand exchange usually terminates after a single round of 180° rotation. However, multiple processive “360° rotation” rounds of strand exchange can be observed, if the recombining sites have nonidentical base pairs at their centers. We propose that a regulatory “gating” mechanism normally blocks multiple rounds of strand exchange and triggers product release after a single round