Developing an in cellulo Carrier-Driven Crystallization System using Cry1Ac from Bacillus thuringiensis

McDade, Kyle Harrison

Developing an in cellulo Carrier-Driven Crystallization System using Cry1Ac from Bacillus thuringiensis

dc.contributor.advisor	Fraser, Marie Elizabeth
dc.contributor.author	McDade, Kyle Harrison
dc.contributor.committeemember	Turner, Raymond Joseph
dc.contributor.committeemember	Hynes, Michael F.
dc.date	2019-11
dc.date.accessioned	2019-09-24T18:40:40Z
dc.date.available	2019-09-24T18:40:40Z
dc.date.issued	2019-09-20
dc.description.abstract	X-ray crystallography is the dominant technique for determining the structures of proteins but suffers from two major issues, the crystallization bottleneck and the phase problem. To address these problems, a solution was proposed in which the Cry1Ac protein, which crystallizes spontaneously in cellulo upon sporulation of Bacillus thuringiensis, is used as a crystallization-carrier – to enable the crystallization of auxiliary proteins. Production of endogenous Cry1Ac was optimized through the characterization of sporulation-inducing media: in comparison to C2 and nutrient sporulation media, G-Tris induced the largest inclusions. Cry1Ac was purified using hexane and ultracentrifugation. Non-mechanical lysis methods were developed to aid purification, including the application of B. thuringiensis cell wall lyase, CwlC. Three fusions of cry1ac with the gene of a reporter, green fluorescent protein (gfp) from Aequorea victoria, were created and integrated within Escherichia coli and B. thuringiensis expression systems. Fusions were designed with GFP at the amino-terminus, at the carboxy-terminus and substituting for the toxin domains of Cry1Ac. Production of the fusion proteins within E. coli generated polarizing and fluorescent inclusions that persisted after sonication. Protein fusions and unfused-Cry1Ac produced recombinantly in E. coli failed to generate bipyramidal inclusions characteristic of Cry1Ac. The carboxy-terminal Cry1Ac-GFP fusion and Cry1Ac exhibited similar solubilities when exposed to high pH and chaotropic agents. Recombinant production of protein fusions and unfused-Cry1Ac within the acrystalloferous B. thuringiensis system was poor: Cells rarely contained inclusions, but inclusions of protein fusions appeared bipyramidal and fluorescent. It is suspected that a reduced rate of sporulation caused by the loss of plasmids pHT8, pAW63, and pHT73 resulted in reduced expression. This issue prohibited inclusion purification and further characterization.	en_US
dc.identifier.citation	McDade, K. H. (2019). Developing an in cellulo Carrier-Driven Crystallization System using Cry1Ac from Bacillus thuringiensis (Master's thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca.	en_US
dc.identifier.doi	http://dx.doi.org/10.11575/PRISM/37114
dc.identifier.uri	http://hdl.handle.net/1880/111052
dc.language.iso	eng	en_US
dc.publisher.faculty	Science	en_US
dc.publisher.institution	University of Calgary	en
dc.rights	University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission.	en_US
dc.subject	Cry1Ac	en_US
dc.subject	Bacillus thringiensis	en_US
dc.subject	carrier-driven crystallization	en_US
dc.subject	in cellulo crystallization	en_US
dc.subject	CwlC	en_US
dc.subject.classification	Biochemistry	en_US
dc.title	Developing an in cellulo Carrier-Driven Crystallization System using Cry1Ac from Bacillus thuringiensis	en_US
dc.type	master thesis	en_US
thesis.degree.discipline	Biological Sciences	en_US
thesis.degree.grantor	University of Calgary	en_US
thesis.degree.name	Master of Science (MSc)	en_US
ucalgary.item.requestcopy	true	en_US