Transposon mediated genetic modification of gram-positive bacteria.

Type of content
Theses / Dissertations
Publisher's DOI/URI
Thesis discipline
Biochemistry
Degree name
Master of Science
Publisher
Journal Title
Journal ISSN
Volume Title
Language
English
Date
2022
Authors
Stocks, Christian F.
Abstract

Genetic modification is vital for modern microbial research. In this thesis, genetic modification explored in bacteria with the use of plasmid vectors that contain modified transposons. Transformation of Gram-positive bacteria was explored through addition of the Himar1 transposon to the previously constructed chromatic bacteria toolbox. Conjugation was then used to insert the Himar-based plasmids into a range of Gram-positive and Gram-negative bacteria. Results of this showed that the Himar-based plasmids could not be used to produce insertion mutants of any Gram-positive strains. However, a handful of Gram-negative strains could be tagged using these plasmids including strains not tagged with the original chromatic bacteria toolbox. Following production of the Himar-based plasmids, transformation protocols were optimised in the Gram-positive strain Arthrobacter sp. Leaf145. Transformation allows for greater amounts of plasmid insertions than conjugation and would increase the number of insertion mutants produced the Himar-based plasmids. However, even after successful optimisation of transformation, insertion mutants were not observed with the Himar-based plasmids in Arthrobacter sp. Leaf145. During transformation optimisation, Tn1409- based plasmids were capable of producing insertion mutants in Arthrobacter sp. Leaf145. Therefore, the use of these plasmids was further explored for native protein over-expression in Arthrobacter sp. Leaf145. Along with genetic analysis of Arthrobacter sp. Leaf145, new Tn1409-based plasmids were constructed which allowed for expression of new genes in Arthrobacter sp. Leaf145. Due to time constraints, the plasmids that would allow for homologous protein over-expression in Arthrobacter sp. Leaf145 could not be tested. However, these are available for use in future research.

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