Thesis

Investigation of novel genetic engineering techniques for Sporosarcina pasteurii bacteria

Creator
Rights statement
Awarding institution
  • University of Strathclyde
Date of award
  • 2022
Thesis identifier
  • T16464
Person Identifier (Local)
  • 202165602
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • In response to the massive carbon footprint associated with the cement industry, much attention has been given to the development of more sustainable alternative materials for use in the construction and ground engineering sectors. The natural biomineralization process exhibited by microbial organisms, particularly by the Sporosarcina pasteurii (S. pasteurii) soil bacteria, holds much potential for development of a lower carbon alternative to cement. The natural ureolytic metabolic pathway of these bacteria is catalyzed by the urease enzyme and in the presence of a calcium source promotes the precipitation of calcium carbonate. Effective implementation of this microbially induced calcium carbonate precipitation (MICP) grouting technology requires optimization to reduce the volume of bacteria/reagents required and increased control over mineral precipitation. Previous studies have experimented with MICP injection strategies and genetic engineering methods that tailor the urease enzyme activity. To date, genetic engineering methods have only used the well understood and commonly genetically manipulated Escherichia coli (E. coli), Pseudomona aeruginosa (P. aeruginosa) and Bacillus subtilis (B. subtilis) bacterial strains instead of S. pasteurii directly. This study aims to develop novel genetic modification methods for the S. pasteurii bacteria via experimentation with published protocol for E. coli and B. subtilis bacteria. In the first phase of this study, expression of synthetic fluorescent coloured proteins was attempted via electroporation and heat shocking transformation methods. Successful transformations and expressions were achieved in the E. coli and B. subtilis bacteria while inconclusive results suggest partially successful transformation of synthetic DNA into the S. pasteurii bacteria. Within these experiments it was established that S. pasteurii has a natural resistance to ampicillin antibiotic and therefore the use of an ampicillin resistance selective gene in transformation methods is not effective. The second phase of this study used synthetically fluorescent bacteria in batch experiments to elucidate the dynamics of calcite crystal nucleation in co-cultures of ureolytic and non-ureolytic cells. Results of the batch experiments indicate higher precipitation rates in co-cultured samples of S. pasteurii and B. subtilis. Indeed, this is supported by microscope analysis of the microfluidic cell experiments which indicates a close association of crystal growth with non-ureolytic cells, supporting the theory that these cells can act as additional nucleation sites.
Advisor / supervisor
  • El Mountassir, Grainne
  • Knapp, Charles
Resource Type
Note
  • This thesis is restricted to Strathclyde users only.
DOI
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