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Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.

View Article: PubMed Central - PubMed

ABSTRACT

Background: Geobacillus thermoglucosidasius is a thermophilic, natural ethanol producer and a potential candidate for commercial bioethanol production. Previously, G. thermoglucosidasius has been genetically modified to create an industrially-relevant ethanol production strain. However, creating chromosomal integrations and deletions in Geobacillus spp. is laborious. Here we describe a new technique to create marker-less mutations in Geobacillus utilising a novel homologous recombination process.

Results: Our technique incorporates counter-selection using β-glucosidase and the synthetic substrate X-Glu, in combination with a two-step homologous recombination process where the first step is a selectable double-crossover event that deletes the target gene. We demonstrate how we have utilised this technique to delete two components of the proteinaceous shell of the Geobacillus propanediol-utilization microcompartment, and simultaneously introduce an oxygen-sensitive promoter in front of the remaining shell-component genes and confirm its functional incorporation.

Conclusion: The selectable deletion of the target gene in the first step of our process prevents re-creation of wild-type which can occur in most homologous recombination techniques, circumventing the need for PCR screening to identify mutants. Our new technique therefore offers a faster, more efficient method of creating mutants in Geobacillus.

No MeSH data available.


Colony PCR to confirm the first crossover event. Following re-streaking, five colonies were selected from agar with kanamycin and X-Glu, boiled in 20 µl milliQ water and PCR amplified with Bgl_F and Bgl_R. Following a double-crossover, the plasmid bgl gene would not be present. Lane 1 Biolabs 1 Kb ladder, lane 2 the 1.1 kb control bgl PCR product from unintegrated TM242 pUCG3.8Bgl-pdu, lanes 3–7 no PCR product from colonies selected on kanamycin X-Glu plates
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Fig2: Colony PCR to confirm the first crossover event. Following re-streaking, five colonies were selected from agar with kanamycin and X-Glu, boiled in 20 µl milliQ water and PCR amplified with Bgl_F and Bgl_R. Following a double-crossover, the plasmid bgl gene would not be present. Lane 1 Biolabs 1 Kb ladder, lane 2 the 1.1 kb control bgl PCR product from unintegrated TM242 pUCG3.8Bgl-pdu, lanes 3–7 no PCR product from colonies selected on kanamycin X-Glu plates

Mentions: The Bgl/X-Glu counter-selection system was implemented to delete the shell proteins of the Pdu MCP. This was undertaken to produce enzymes normally present within the Pdu MCP in the cytoplasm, thus removing the selectivity of the MCP shell [18]. The shell genes pduA and B were replaced with an ldh promoter to eliminate native regulation and upregulate the metabolic enzymes in the rest of the pdu operon, downstream of pduB. The plasmid pUCG3.8Bgl-pdu was electroporated into G. thermoglucosidasius TM242. Figure 1 shows the integration and deletion strategy. The first integration event was selected by growth on SPYNG medium containing kanamycin and 1 mg/ml X-Glu, which should counter-select for the un-integrated plasmid. If the incorporation of bgl gene into the chromosome from a single crossover event increased the production of the toxic indigoid dye to a level that was non-permissive, growth should only occur following a double crossover event incorporating the kanamycin resistance gene but not bgl. Although preliminary tests had used a high copy number plasmid, it was anticipated that use of the strong promoter rpsL would also result in sufficiently high bgl expression levels after chromosomal integration to confer toxicity to X-Glu at the concentration tolerated by the WT strain. Approximately 150 colonies were observed on the X-Glu and kanamycin plates, and were assumed to contain double-crossover integrations. A single, large colony was selected, re-streaked onto X-Glu and kanamycin agar for purification, and incubated overnight. Five colonies were selected and colony PCR was used to confirm the absence of the plasmid backbone, confirming that all colonies had lost the bgl gene, but as they were kanamycin resistant, presumably contained the desired double crossover integrations (Fig. 2). Therefore, our assumption that a single crossover incorporating a single copy of bgl expressed from a strong promoter would be toxic was confirmed. One of the colonies containing a double crossover integration was selected, passaged four times in SPYNG media containing no antibiotic to encourage the second crossover recombination, and then plated onto agar with no antibiotic added. Three hundred and fifty colonies were selected, replica-plated onto agar with and without kanamycin and incubated overnight. Sensitive colonies would occur following a second crossover event, creating a marker-less chromosomal target gene deletion mutant. Five colonies were found to be sensitive to kanamycin, and colony PCR confirmed that these sensitive colonies were indeed deletion mutants (Fig. 3).Fig. 1


Development of an efficient technique for gene deletion and allelic exchange in Geobacillus spp.
Colony PCR to confirm the first crossover event. Following re-streaking, five colonies were selected from agar with kanamycin and X-Glu, boiled in 20 µl milliQ water and PCR amplified with Bgl_F and Bgl_R. Following a double-crossover, the plasmid bgl gene would not be present. Lane 1 Biolabs 1 Kb ladder, lane 2 the 1.1 kb control bgl PCR product from unintegrated TM242 pUCG3.8Bgl-pdu, lanes 3–7 no PCR product from colonies selected on kanamycin X-Glu plates
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC5382374&req=5

Fig2: Colony PCR to confirm the first crossover event. Following re-streaking, five colonies were selected from agar with kanamycin and X-Glu, boiled in 20 µl milliQ water and PCR amplified with Bgl_F and Bgl_R. Following a double-crossover, the plasmid bgl gene would not be present. Lane 1 Biolabs 1 Kb ladder, lane 2 the 1.1 kb control bgl PCR product from unintegrated TM242 pUCG3.8Bgl-pdu, lanes 3–7 no PCR product from colonies selected on kanamycin X-Glu plates
Mentions: The Bgl/X-Glu counter-selection system was implemented to delete the shell proteins of the Pdu MCP. This was undertaken to produce enzymes normally present within the Pdu MCP in the cytoplasm, thus removing the selectivity of the MCP shell [18]. The shell genes pduA and B were replaced with an ldh promoter to eliminate native regulation and upregulate the metabolic enzymes in the rest of the pdu operon, downstream of pduB. The plasmid pUCG3.8Bgl-pdu was electroporated into G. thermoglucosidasius TM242. Figure 1 shows the integration and deletion strategy. The first integration event was selected by growth on SPYNG medium containing kanamycin and 1 mg/ml X-Glu, which should counter-select for the un-integrated plasmid. If the incorporation of bgl gene into the chromosome from a single crossover event increased the production of the toxic indigoid dye to a level that was non-permissive, growth should only occur following a double crossover event incorporating the kanamycin resistance gene but not bgl. Although preliminary tests had used a high copy number plasmid, it was anticipated that use of the strong promoter rpsL would also result in sufficiently high bgl expression levels after chromosomal integration to confer toxicity to X-Glu at the concentration tolerated by the WT strain. Approximately 150 colonies were observed on the X-Glu and kanamycin plates, and were assumed to contain double-crossover integrations. A single, large colony was selected, re-streaked onto X-Glu and kanamycin agar for purification, and incubated overnight. Five colonies were selected and colony PCR was used to confirm the absence of the plasmid backbone, confirming that all colonies had lost the bgl gene, but as they were kanamycin resistant, presumably contained the desired double crossover integrations (Fig. 2). Therefore, our assumption that a single crossover incorporating a single copy of bgl expressed from a strong promoter would be toxic was confirmed. One of the colonies containing a double crossover integration was selected, passaged four times in SPYNG media containing no antibiotic to encourage the second crossover recombination, and then plated onto agar with no antibiotic added. Three hundred and fifty colonies were selected, replica-plated onto agar with and without kanamycin and incubated overnight. Sensitive colonies would occur following a second crossover event, creating a marker-less chromosomal target gene deletion mutant. Five colonies were found to be sensitive to kanamycin, and colony PCR confirmed that these sensitive colonies were indeed deletion mutants (Fig. 3).Fig. 1

View Article: PubMed Central - PubMed

ABSTRACT

Background: Geobacillus thermoglucosidasius is a thermophilic, natural ethanol producer and a potential candidate for commercial bioethanol production. Previously, G. thermoglucosidasius has been genetically modified to create an industrially-relevant ethanol production strain. However, creating chromosomal integrations and deletions in Geobacillus spp. is laborious. Here we describe a new technique to create marker-less mutations in Geobacillus utilising a novel homologous recombination process.

Results: Our technique incorporates counter-selection using β-glucosidase and the synthetic substrate X-Glu, in combination with a two-step homologous recombination process where the first step is a selectable double-crossover event that deletes the target gene. We demonstrate how we have utilised this technique to delete two components of the proteinaceous shell of the Geobacillus propanediol-utilization microcompartment, and simultaneously introduce an oxygen-sensitive promoter in front of the remaining shell-component genes and confirm its functional incorporation.

Conclusion: The selectable deletion of the target gene in the first step of our process prevents re-creation of wild-type which can occur in most homologous recombination techniques, circumventing the need for PCR screening to identify mutants. Our new technique therefore offers a faster, more efficient method of creating mutants in Geobacillus.

No MeSH data available.