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Construction of an Inducible Adhesion Circuit for the Colonization of Bacillus megaterium Sensor Cells

Larsson, Elin LU (2019) KBKM05 20191
Pure and Applied Biochemistry
Computational Chemistry
Abstract
Many of our freshwater resources are contaminated with pollutants such as heavy metals and organophosphates, and therefore the demand for reliable and efficient methods for detection and remediation of such pollutants is increasing. One widely used method for remediation of water is through the use of engineered communities of microbes that have the ability to degrade or make the contaminating compounds less harmful. These microbial communities often rely on biofilm formation as a means of survival. When building a system with sensing or remediating cells, immobilizing these cells in a biofilm is beneficial because it increases the lifetime of the community and retains them at the site of contamination. We want to build a community of... (More)
Many of our freshwater resources are contaminated with pollutants such as heavy metals and organophosphates, and therefore the demand for reliable and efficient methods for detection and remediation of such pollutants is increasing. One widely used method for remediation of water is through the use of engineered communities of microbes that have the ability to degrade or make the contaminating compounds less harmful. These microbial communities often rely on biofilm formation as a means of survival. When building a system with sensing or remediating cells, immobilizing these cells in a biofilm is beneficial because it increases the lifetime of the community and retains them at the site of contamination. We want to build a community of sensing and remediation cells with an inducible system for biofilm formation. One strategy to induce biofilm formation is through the expression of amyloid protein fibers that promote cell-cell adhesion and cluster formation. for microbial-mediated environmental sensing and remediation purposes, a robust species, adapted for environmental survival, is desirable. For this reason we have chosen Bacillus megaterium, a ubiquitous soil bacterium, as a chassis for environmental sensing and remediation. Here, as proof-of-concept, we engineered a xylose-inducible system for the expression of different amyloid proteins in B. megaterium. Using this system, we show the expression and secretion of two amyloid proteins, the metabolic burden incurred by their expression, and suggest an assay to evaluate the degree of retention the expression of these proteins adds to cells on a matrix. We found that when the engineered plasmids containing csgA and tasA genes were transformed into B. megaterium, the cells were successfully able to express these amyloid proteins. However, only TasA was exported, whereas CsgA was accumulated inside the cells. Furthermore, expression of TasA results in only a small growth penalty. (Less)
Popular Abstract (Swedish)
På grund av människans aktiviteter har många av världens vattenresurser förorenats. Dricksvatten förorenat av arsenik, en tungmetall som kan orsaka akut förgiftning och en uppsjö av sjukdomar, blir allt vanligare världen över. I och med detta ökar behovet av sätt att detektera och oskadliggöra tungmetaller och även olika typer av skadliga kemikalier i våra vatten.

Tänk dig ett system där bakterier kan känna av närvaron av en farlig kemikalie, samlas runt den och börja bryta ner den. Tanken bakom detta forskningsprojekt var att använda bakteriearten Bacillus megaterium, en jättebakterie som kan bli ungefär hundra gånger så stor som E. coli, för att åstadkomma en viktig del av det tänkta systemet. För att kunna skydda sig själva och på... (More)
På grund av människans aktiviteter har många av världens vattenresurser förorenats. Dricksvatten förorenat av arsenik, en tungmetall som kan orsaka akut förgiftning och en uppsjö av sjukdomar, blir allt vanligare världen över. I och med detta ökar behovet av sätt att detektera och oskadliggöra tungmetaller och även olika typer av skadliga kemikalier i våra vatten.

Tänk dig ett system där bakterier kan känna av närvaron av en farlig kemikalie, samlas runt den och börja bryta ner den. Tanken bakom detta forskningsprojekt var att använda bakteriearten Bacillus megaterium, en jättebakterie som kan bli ungefär hundra gånger så stor som E. coli, för att åstadkomma en viktig del av det tänkta systemet. För att kunna skydda sig själva och på så sätt ha större chans att överleva, bildar bakteriekulturer ofta så kallade biofilmer. En biofilm består ofta av många olika bakteriearter och ämnen, till exempel polysackarider, som får dem att hålla ihop i en struktur. Den skyddande strukturen bidrar till att cellerna blir mer skyddade mot yttre påverkan i mediet, till exempel av antimikrobiella ämnen, pH-förändringar och mekanisk stress. I detta projekt designade vi en plasmid med gener för uttryck av proteiner som kan initiera formationen av en biofilm. När inducern xylos tillsätts till tillväxtmediet, börjar ett protein uttryckas och produceras för att sedan exporteras till cellens yta. Där kan det bilda aggregat med proteiner på andra cellers ytor och på så sätt bilda kluster av celler. I den slutgiltliga applikationen kan xylos bytas ut mot en annan inducer, till exempel arsenik eller andra tungmetaller som kvicksilver och bly. På så sätt kan tungmetaller i vattnet initiera någon form av respons hos bakterierna i biosensorn.

I denna studie uttryckte vi amyloidproteiner i B. megaterium i ett försök att kontrollera cellernas förmåga att bilda kluster som sedan skulle kunna bli en del av en syntetisk biofilm. Vi fann att proteinet CsgA fastnar inuti cellerna, medan proteinet TasA transporteras utanför cellerna. (Less)
Please use this url to cite or link to this publication:
author
Larsson, Elin LU
supervisor
organization
course
KBKM05 20191
year
type
H2 - Master's Degree (Two Years)
subject
keywords
Bacillus megaterium, amyloids, adhesion, arsenic, applied biochemistry
language
English
id
8984963
date added to LUP
2022-08-01 12:22:19
date last changed
2022-08-01 12:22:19
@misc{8984963,
  abstract     = {{Many of our freshwater resources are contaminated with pollutants such as heavy metals and organophosphates, and therefore the demand for reliable and efficient methods for detection and remediation of such pollutants is increasing. One widely used method for remediation of water is through the use of engineered communities of microbes that have the ability to degrade or make the contaminating compounds less harmful. These microbial communities often rely on biofilm formation as a means of survival. When building a system with sensing or remediating cells, immobilizing these cells in a biofilm is beneficial because it increases the lifetime of the community and retains them at the site of contamination. We want to build a community of sensing and remediation cells with an inducible system for biofilm formation. One strategy to induce biofilm formation is through the expression of amyloid protein fibers that promote cell-cell adhesion and cluster formation. for microbial-mediated environmental sensing and remediation purposes, a robust species, adapted for environmental survival, is desirable. For this reason we have chosen Bacillus megaterium, a ubiquitous soil bacterium, as a chassis for environmental sensing and remediation. Here, as proof-of-concept, we engineered a xylose-inducible system for the expression of different amyloid proteins in B. megaterium. Using this system, we show the expression and secretion of two amyloid proteins, the metabolic burden incurred by their expression, and suggest an assay to evaluate the degree of retention the expression of these proteins adds to cells on a matrix. We found that when the engineered plasmids containing csgA and tasA genes were transformed into B. megaterium, the cells were successfully able to express these amyloid proteins. However, only TasA was exported, whereas CsgA was accumulated inside the cells. Furthermore, expression of TasA results in only a small growth penalty.}},
  author       = {{Larsson, Elin}},
  language     = {{eng}},
  note         = {{Student Paper}},
  title        = {{Construction of an Inducible Adhesion Circuit for the Colonization of Bacillus megaterium Sensor Cells}},
  year         = {{2019}},
}