Presentation Title

Subcloning of the ISGA 1218 NtaA Gene from Gordonia sp. Strain NB4-1Y for Large Scale Protein Production

Presenter Information

Connor Milton-Wood

Location

House of Learning Library, 3rd floor

Start Date

18-3-2016 12:00 PM

End Date

18-3-2016 6:00 PM

Abstract

Compounds known as sulfonated per- and poly-fluoroalkyl substances (PFAS) have emerged as environmental contaminants of concern due to their ability to bioaccumulate, transport over long distances, and cause toxicity. One such PFAS, known to be used in the telomerisation-based aqueous film-forming foams used in the wildfire fighting and aviation industries, is 6:2 fluorotelomer sulfonate (FTS), a compound previously recognized as a surface and groundwater contaminant that can bioaccumulate in fish livers. The soil bacterium Gordonia sp. strain NB4-1Y has been observed to utilize 6:2 FTS as a source of sulfur, possibly through the activity of two nitrilotriacetate (NtaA) monooxygenase enzymes, presenting an opportunity for the bioremediation of sulfonated PFAS. In a previous study, one of the genes encoding an NtaA enzyme from the NB4-1Y genome, ISGA 1218, was cloned into a pBluescript (pBS) plasmid vector and transferred into Escherichia coli DH5ɑ. This study aims to transfer the ISGA 1218 gene from the pBS vector into a pMAL expression vector to facilitate large scale production and purification of the ISGA 1218 enzyme for study. The pMAL expression vector introduces a maltose-binding protein onto the N-terminus of the target protein, allowing for protein purification by amylose affinity chromatography. The purified enzyme will subsequently be tested in vitro for activity against 6:2 FTS to assess the validity of its proposed role in NB4-1Y FTS metabolism.

Department

Biological Sciences

Faculty Advisor

Jonathan Van Hamme

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Mar 18th, 12:00 PM Mar 18th, 6:00 PM

Subcloning of the ISGA 1218 NtaA Gene from Gordonia sp. Strain NB4-1Y for Large Scale Protein Production

House of Learning Library, 3rd floor

Compounds known as sulfonated per- and poly-fluoroalkyl substances (PFAS) have emerged as environmental contaminants of concern due to their ability to bioaccumulate, transport over long distances, and cause toxicity. One such PFAS, known to be used in the telomerisation-based aqueous film-forming foams used in the wildfire fighting and aviation industries, is 6:2 fluorotelomer sulfonate (FTS), a compound previously recognized as a surface and groundwater contaminant that can bioaccumulate in fish livers. The soil bacterium Gordonia sp. strain NB4-1Y has been observed to utilize 6:2 FTS as a source of sulfur, possibly through the activity of two nitrilotriacetate (NtaA) monooxygenase enzymes, presenting an opportunity for the bioremediation of sulfonated PFAS. In a previous study, one of the genes encoding an NtaA enzyme from the NB4-1Y genome, ISGA 1218, was cloned into a pBluescript (pBS) plasmid vector and transferred into Escherichia coli DH5ɑ. This study aims to transfer the ISGA 1218 gene from the pBS vector into a pMAL expression vector to facilitate large scale production and purification of the ISGA 1218 enzyme for study. The pMAL expression vector introduces a maltose-binding protein onto the N-terminus of the target protein, allowing for protein purification by amylose affinity chromatography. The purified enzyme will subsequently be tested in vitro for activity against 6:2 FTS to assess the validity of its proposed role in NB4-1Y FTS metabolism.