https://ogma.newcastle.edu.au/vital/access/ /manager/Index ${session.getAttribute("locale")} 5 https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:45135 Bifidobacterium are notoriously recalcitrant to genetic manipulation due to their extensive and variable repertoire of Restriction-Modification (R-M) systems. Non-replicating plasmids are currently employed to achieve insertional mutagenesis in Bifidobacterium. One of the limitations of using such insertion vectors is the presence within their sequence of various restriction sites, making them sensitive to the activity of endogenous restriction endonucleases encoded by the target strain. For this reason, vectors have been developed with the aim of methylating and protecting the vector using a methylase-positive Escherichia coli strain, in some cases containing a cloned bifidobacterial methylase. Here, we present a mutagenesis approach based on a modified and synthetically produced version of the suicide vector pORI28 (named pFREM28), where all known restriction sites targeted by Bifidobacterium breve R-M systems were removed by base substitution (thus preserving the codon usage). After validating the integrity of the erythromycin marker, the vector was successfully employed to target an a-galactosidase gene responsible for raffinose metabolism, an alcohol dehydrogenase gene responsible for mannitol utilization and a gene encoding a priming glycosyltransferase responsible for exopolysaccharides (EPS) production in B. breve. The advantage of using this modified approach is the reduction of the amount of time, effort and resources required to generate site-directed mutants in B. breve and a similar approach may be employed to target other (bifido)bacterial species.]]> Wed 26 Oct 2022 13:12:44 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:2506 Wed 11 Apr 2018 16:56:05 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:12630 Wed 11 Apr 2018 10:07:15 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:4896 Wed 11 Apr 2018 09:40:29 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:34763 Wed 10 Nov 2021 15:04:10 AEDT ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:43143 Tue 13 Sep 2022 15:21:34 AEST ]]> https://ogma.newcastle.edu.au/vital/access/ /manager/Repository/uon:48515 mcyA-J. The recent establishment of a cyanotoxin heterologous expression system in Escherichia coli has provided the means to study microcystin biosynthesis in a genetically tractable, rapidly growing host. Using this system, we demonstrate that deletion of the ABC-transporter, mcyH, and dehydrogenase, mcyI, abolishes microcystin production, while deletion of the O-methyltransferase, mcyJ, results in the production of the demethylated (DM) toxin [D-Asp³ , DMAdda ⁵]microcystin-LR. Both methylated and DM toxin variants were heterologously produced at high titers and efficiently exported into the extracellular medium, enabling easy purification. The results show that the mcy gene cluster can be engineered in E. coli to study the function of its individual components and direct the synthesis of particular microcystin variants. This technology could potentially be applied to other natural products of ecological and biomedical significance.]]> Mon 20 Mar 2023 16:46:36 AEDT ]]>