Generation of Pseudomonas mutants for investigating a model mixed species biofilm
Mixed-species microbial biofilms are the predominant form of biofilms in nature, however, majority of studies on biofilms have focused on single-species biofilms. Unique interspecies interactions could shape the biofilm community structure and functions in a manner that is not represented in single-...
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sg-ntu-dr.10356-746242023-02-28T18:01:43Z Generation of Pseudomonas mutants for investigating a model mixed species biofilm Neo, Desmond Scott Rice School of Biological Sciences DRNTU::Science Mixed-species microbial biofilms are the predominant form of biofilms in nature, however, majority of studies on biofilms have focused on single-species biofilms. Unique interspecies interactions could shape the biofilm community structure and functions in a manner that is not represented in single-species biofilms. Thus, a model mixed-species biofilm comprised of Klebsiella pneumoniae (KP-1), Pseudomonas aeruginosa (PAO1) and Pseudomonas protegens (Pf-5) was selected to investigate the differentially expressed genes between single-species and mixed-species biofilms to identify key genes within Pf-5 involved in overall biofilm structure via mutagenesis studies. This study involved the fluorescent tagging of PAO1 via specific insertion mutagenesis involving a Tn7 transposon. Deletion mutagenesis of differentially expressed genes in Pf-5 were performed, with PFL_5251 as the target gene to develop a reliable method for deletion mutagenesis in Pf-5 where multiple key methods were identified at each stage. Both electroporation and conjugation were evaluated for the introduction of pK18 assembled with deletion construct into Pf-5. Both failed to select for transformants and transconjugants until the antibiotic concentrations were optimized. PFL_5251 mutants were generated and confirmed for Pf-5 via sequence alignment. This paved the way for further analysis with bioimaging techniques to help improve understanding and determination of genes important in mixed-species biofilms. Bachelor of Science in Biological Sciences 2018-05-22T06:49:09Z 2018-05-22T06:49:09Z 2018 Final Year Project (FYP) http://hdl.handle.net/10356/74624 en Nanyang Technological University 49 p. application/pdf |
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DRNTU::Science Neo, Desmond Generation of Pseudomonas mutants for investigating a model mixed species biofilm |
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Mixed-species microbial biofilms are the predominant form of biofilms in nature, however, majority of studies on biofilms have focused on single-species biofilms. Unique interspecies interactions could shape the biofilm community structure and functions in a manner that is not represented in single-species biofilms. Thus, a model mixed-species biofilm comprised of Klebsiella pneumoniae (KP-1), Pseudomonas aeruginosa (PAO1) and Pseudomonas protegens (Pf-5) was selected to investigate the differentially expressed genes between single-species and mixed-species biofilms to identify key genes within Pf-5 involved in overall biofilm structure via mutagenesis studies. This study involved the fluorescent tagging of PAO1 via specific insertion mutagenesis involving a Tn7 transposon. Deletion mutagenesis of differentially expressed genes in Pf-5 were performed, with PFL_5251 as the target gene to develop a reliable method for deletion mutagenesis in Pf-5 where multiple key methods were identified at each stage. Both electroporation and conjugation were evaluated for the introduction of pK18 assembled with deletion construct into Pf-5. Both failed to select for transformants and transconjugants until the antibiotic concentrations were optimized. PFL_5251 mutants were generated and confirmed for Pf-5 via sequence alignment. This paved the way for further analysis with bioimaging techniques to help improve understanding and determination of genes important in mixed-species biofilms. |
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Scott Rice |
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Scott Rice Neo, Desmond |
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Final Year Project |
author |
Neo, Desmond |
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Neo, Desmond |
title |
Generation of Pseudomonas mutants for investigating a model mixed species biofilm |
title_short |
Generation of Pseudomonas mutants for investigating a model mixed species biofilm |
title_full |
Generation of Pseudomonas mutants for investigating a model mixed species biofilm |
title_fullStr |
Generation of Pseudomonas mutants for investigating a model mixed species biofilm |
title_full_unstemmed |
Generation of Pseudomonas mutants for investigating a model mixed species biofilm |
title_sort |
generation of pseudomonas mutants for investigating a model mixed species biofilm |
publishDate |
2018 |
url |
http://hdl.handle.net/10356/74624 |
_version_ |
1759853373655351296 |