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Source: https://theses.gla.ac.uk/30585/ Parent: https://theses.gla.ac.uk/view/subjects/QH345.html

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Serine integrase-based “landing pad” systems for chromosomal integrations of heterologous genes in Escherichia coli

Clubbe, Richard (2018) Serine integrase-based “landing pad” systems for chromosomal integrations of heterologous genes in Escherichia coli. PhD thesis, University of Glasgow.

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Printed Thesis Information: https://eleanor.lib.gla.ac.uk/record=b3327845

Abstract

Due to its long history of industrial applications, extensively studied biology and rapid growth rates, Escherichia coli is an important cell factory for metabolic engineering and an increasingly popular chassis organism for synthetic biology. Serine integrases, which function naturally to incorporate bacteriophage DNA into the host genome, represent a powerful and flexible tool for the genomic integration of heterologous genes or pathways into host organisms.\ In the system described here, serine integrase enzymes are used to recombine two pairs of non-identical attachment sites, located on a chromosomally-integrated “landing pad” (LP) and an introduced “donor cassette”, resulting in cassette exchange of a transgene into a known locus on the E. coli genome. In this work, a landing pad was designed for φC31 integrase, consisting of two φC31 attB sites, both with differing central dinucleotide pairs, in a head-to-head orientation flanking an erythromycin resistance marker gene. This φC31 LP was inserted into the E. coli genome, replacing the non-essential pepA gene, by recombineering. Cassette exchange experiments were then performed, in which a temperature-sensitive plasmid carrying a donor cassette consisting of corresponding attP sites to the φC31 LP attB sites flanking an antibiotic resistance marker was transformed under the inducible expression of φC31 integrase.\ Serine integrases recombine their target sites in a highly unidirectional reaction. The reverse excision reaction, between integration product sites attL and attR, can only proceed with the addition of the integrase-associated recombinational directionality factor (RDF). Using in vitro recombination assays, TG1 integrase was shown to have very highly controlled directionality. Addition of the TG1 RDF gp25 strongly activates attL-attR recombination, taking recombination activity from a background level of 9% to 72% recombination of the substrate. TG1 gp25 also strongly inhibits attP-attB recombination, decreasing recombination from 77% to 8%. This demonstrates its potential as a highly controllable recombinase for synthetic biology. This work also investigated cross-reactivity of integrases and their non-cognate RDFs, showing that the TG1 RDF gp25 is able to activate φC31 integrase attL- attR recombination, however does not similarly inhibit φC31 attP-attB.

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