A motif in the C-terminal domain of ϕC31 integrase controls the directionality of recombination

Bacteriophage ϕC31 encodes an integrase, which acts on the phage and host attachment sites, attP and attB, to form an integrated prophage flanked by attL and attR. In the absence of accessory factors, ϕC31 integrase cannot catalyse attL x attR recombination to excise the prophage. To understand the mechanism of directionality, mutant integrases were characterized that were active in excision. A hyperactive integrase, Int E449K, gained the ability to catalyse attL x attR, attL x attL and attR x attR recombination whilst retaining the ability to recombine attP x attB. A catalytically defective derivative of this mutant, Int S12A, E449K, could form stable complexes with attP/attB, attL/attR, attL/attL and attR/attR under conditions where Int S12A only complexed with attP/attB. Further analysis of the Int E449K-attL/attR synaptic events revealed a preference for one of the two predicted synapse structures with different orientations of the attL/attR sites. Several amino acid substitutions conferring hyperactivity, including E449K, were localized to one face of a predicted coiled-coil motif in the C-terminal domain. This work shows that a motif in the C-terminal domain of ϕC31 integrase controls the formation of the synaptic interface in both integration and excision, possibly through a direct role in protein–protein interactions

New Applications for Phage Integrases

Within the last twenty-five years bacteriophage integrases have rapidly risen to prominence as genetic tools for a wide range of applications from basic cloning to genome engineering. Serine integrases such as that from ϕC31 and its relatives have found an especially wide-range of applications within diverse micro-organisms right through to multi-cellular eukaryotes. Here we review the mechanisms of the two major families of integrases, the tyrosine and serine integrases, and the advantages and disadvantages of each type as they are applied in genome engineering and synthetic biology. In particular, we focus on the new areas of metabolic pathway construction and optimisation, bio-computing, heterologous expression and multiplexed assembly techniques. Integrases are versatile and efficient tools that can be used in conjunction with the various extant molecular biology tools to streamline the synthetic biology production line

Defining a Roadmap for Economically Efficient Digital Curation – a 4C Project Workshop: Workshops and Tutorials - iPRES 2014 - Melbourne

The 4C Project is tasked with delivering a Roadmap report and it is this drive towards ‘economic efficiency’ in relation to digital curation that will be central to the agenda that it sets out. This workshop is an important opportunity to connect with stakeholders and get input for a critical deliverable of the project. But it is also an opportunity for participants to learn more about the economics of digital curation and to critically assess the efficiency and sustainability of their own services and solutions

Direct Synthesis of Maradolipids and Other Trehalose 6‑Monoesters and 6,6′-Diesters

It was shown that reaction of trehalose with 1 equiv of a fatty acid in pyridine promoted by 1 equiv of the uronium-based coupling agent 2-(1<i>H</i>-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) at room temperature gives a good yield of the primary ester accompanied by small amounts of the diprimary ester using hexanoic, palmitic, and oleic acids as examples. Reactions using 2 equiv of the fatty acids gave the symmetrical diesters. The monoesters were reacted with different fatty acids to give nonsymmetric 6,6′-diesters in very good yields. Compounds synthesized include the most abundant component of the very complex maradolipid mixture, 6-<i>O</i>-(13-methyltetradecanoyl)-6′-<i>O</i>-oleoyltrehalose, and a component potentially present in this mixture, 6-<i>O</i>-(12-methyltetradecanoyl)-6′-<i>O</i>-oleoyltrehalose, a derivative of an ante fatty acid. The C5–C6 rotameric populations of 6-<i>O</i>-monoesters, symmetrical 6,6′-diesters, and 2,6,6′-triesters of fatty acids were calculated from the values of the H5–H6<i>R</i> and H5–H6<i>S</i> coupling constants and found to be similar to those found for glucose. The rotameric populations of the monosubstituted glucose residues in the 2,6,6′-triesters was altered considerably to favor the <i>gt</i> rotamer, presumably because of attraction between the 2- and 6′-fatty acid chains

Birds of Mondulkiri, Cambodia: distribution, status and conservation

Volume: 30Start Page: 66End Page: 7