Dublin Core
Title
Engineered bacteria for propionate-repressed expression of sfGFP and GM-CSF
Subject
616.344
Medicina y salud
Enfermedades inflamatorias del intestino
Escherichia coli
Plásmidos
Description
Tesis (Master of Science in Engineering)--Pontificia Universidad Católica de Chile, 2021
Inflammatory bowel diseases (IBD) are chronic diseases that currently have no cure. Their
causes are multifactorial, but the composition of the gut microbiome is believed to play an
important role in their development. Certain beneficial bacteria in the microbiome are able
to ferment dietary fibers and produce short chain fatty acids (SCFA), such as propionate, that
help protect intestinal integrity and prevent the colonization of pathogens. These are found
in reduced concentrations in people who suffer from IBD. Taking advantage of the ability of
bacteria to colonize the human body, they have been engineered to act as biosensors to detect
specific biomarkers which can be used as complimentary diagnostic tools. They have also
been genetically modified to act as drug delivery vehicles, helping to increase target
specificity and avoid harmful side effects. In this work, we develop a two plasmid system in
E. coli DH5α in which propionate induces the expression of the LacI repressor. In the second
plasmid, the repressor controls the expression of the reporter protein, sfGFP. We observed
that the genetic circuit functions between 0-110 mM of propionate with at least 0.1 mM
generating a response. We then replaced sfGFP with the gene for the cytokine, granulocyte
macrophage-colony stimulating factor (GM-CSF), which was chosen due to its reported
effects in strengthening the intestinal epithelium barrier. After 4 hours of growth and in the
absence of propionate, 11.61 μg/mL of GM-CSF/OD600 were produced, while 8.959 μg/mL
were produced in the presence of 100 mM of the inductor. Nevertheless, the differences in
the concentration of GM-CSF produced were minimal between the two conditions after this
time. Additionally, the concentration of GM-CSF produced rapidly declined after 4 hours,
likely being degraded within the cell. Overall, the reporter strain may function as a
complementary diagnostic tool for quantifying propionate and the therapeutic strain may
work as a delivery vehicle for GM-CSF, although further studies are required to perfect the
sense and respond mechanism.
causes are multifactorial, but the composition of the gut microbiome is believed to play an
important role in their development. Certain beneficial bacteria in the microbiome are able
to ferment dietary fibers and produce short chain fatty acids (SCFA), such as propionate, that
help protect intestinal integrity and prevent the colonization of pathogens. These are found
in reduced concentrations in people who suffer from IBD. Taking advantage of the ability of
bacteria to colonize the human body, they have been engineered to act as biosensors to detect
specific biomarkers which can be used as complimentary diagnostic tools. They have also
been genetically modified to act as drug delivery vehicles, helping to increase target
specificity and avoid harmful side effects. In this work, we develop a two plasmid system in
E. coli DH5α in which propionate induces the expression of the LacI repressor. In the second
plasmid, the repressor controls the expression of the reporter protein, sfGFP. We observed
that the genetic circuit functions between 0-110 mM of propionate with at least 0.1 mM
generating a response. We then replaced sfGFP with the gene for the cytokine, granulocyte
macrophage-colony stimulating factor (GM-CSF), which was chosen due to its reported
effects in strengthening the intestinal epithelium barrier. After 4 hours of growth and in the
absence of propionate, 11.61 μg/mL of GM-CSF/OD600 were produced, while 8.959 μg/mL
were produced in the presence of 100 mM of the inductor. Nevertheless, the differences in
the concentration of GM-CSF produced were minimal between the two conditions after this
time. Additionally, the concentration of GM-CSF produced rapidly declined after 4 hours,
likely being degraded within the cell. Overall, the reporter strain may function as a
complementary diagnostic tool for quantifying propionate and the therapeutic strain may
work as a delivery vehicle for GM-CSF, although further studies are required to perfect the
sense and respond mechanism.
Creator
Barra, María José
Date
2021-04-05T11:47:02Z
2021-04-05T11:47:02Z
2021
Contributor
Garrido Cortés, Daniel
Pontificia Universidad Católica de Chile. Escuela de Ingeniería
Rights
acceso abierto
Format
v, 53, 10 páginas
application/pdf
Language
en
Type
tesis de maestría
Identifier
10.7764/tesisUC/ING/57238
https://doi.org/10.7764/tesisUC/ING/57238
https://repositorio.uc.cl/handle/11534/57238