Difference between revisions of "Part:BBa K2721010"
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==Approach and Result== | ==Approach and Result== | ||
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| + | <h5><h5 style="font-weight: 300 !important; text-align: left; padding-left: 2% !important; width: 93% !important; display: inline-block;"><span style="font-weight: bolder !important;">Results of agarose gel electrophoresis.CsgA is about 530bp.Bands are visible between 500bp and 750bp.The deficiency of band of knock-out strain is as expected.</h5></h5> | ||
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| + | <html><p>SDS-PAGE results:</p> | ||
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<h5><h5 style="font-weight: 300 !important; text-align: left; padding-left: 2% !important; width: 93% !important; display: inline-block;"><span style="font-weight: bolder !important;">Quantitative Congo Red (CR) Binding Assays to verify the successful expression of Curli. (A) a is a wild-type E.coli strain that normally expresses csgA protein. b is the PHL628-△csgA cells (csgA knock out) strain. It can be seen that there is almost no expression of csgA after knocking out. (B) a is the wild type strain, b is the csgA overexpressing strain (a plasmid carrying a csga protein is expressed in wild type E.coli), and c is the csga KO cell. By adding Congo red dye to the bacteria mixture, centrifugation and shaking, we can compare the expression level of curli by observing the color depth of the precipitate. (C) Figure C is an inverted view of the EP tube in Figure B, showing the color of the sediment at the bottom of the centrifuge tube. a – WT. b – csgA KO, c – csgA overexpressing.</h5></h5> | <h5><h5 style="font-weight: 300 !important; text-align: left; padding-left: 2% !important; width: 93% !important; display: inline-block;"><span style="font-weight: bolder !important;">Quantitative Congo Red (CR) Binding Assays to verify the successful expression of Curli. (A) a is a wild-type E.coli strain that normally expresses csgA protein. b is the PHL628-△csgA cells (csgA knock out) strain. It can be seen that there is almost no expression of csgA after knocking out. (B) a is the wild type strain, b is the csgA overexpressing strain (a plasmid carrying a csga protein is expressed in wild type E.coli), and c is the csga KO cell. By adding Congo red dye to the bacteria mixture, centrifugation and shaking, we can compare the expression level of curli by observing the color depth of the precipitate. (C) Figure C is an inverted view of the EP tube in Figure B, showing the color of the sediment at the bottom of the centrifuge tube. a – WT. b – csgA KO, c – csgA overexpressing.</h5></h5> | ||
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Latest revision as of 18:31, 17 October 2018
the gene of csgA
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Usage and Biology
CsgA is the monomer of curli, which is an important component of excellular matrix of Enterobacteriaceae. Curli fibers are involved in adhesion to surfaces, cell aggregation, and biofilm formation, they are also used for studies of amyloid fiber formation in human diseases, such as Alzheimer’s, Huntington’s, and prion diseases.
As an important component of excellular matrix and biofilms, modified csgA provides us many potentials to remould biofilm into a brand-new functional biology matrix. We can tack many fancy characteristics to biofilm and modified bacteria. Team ZJU-CHINA 2018 made use of the design of cell-surface display and modified the csgA protein by fusing spytag to the N-terminal, making it a dorable platform to form enzyme complex and increasd the electrical conductivity because of the histag in the C-terminal.Actually, the cell-surface display has many potential applications, including biodegradation,live vaccine development, peptide library screening, bioconversion using whole cell biocatalyst and bioadsorption.
As the excellular matrix of bacteria, the curli can also interact with intracellular reactions, and function as a detector of environment or messenger between extracellular and intracellular. For example, the modified curli can be used as a membrane-based bisensor for pathogen, heavy metal and other environment pollutants.
Approach and Result
agarose gel electrophoresis:
Results of agarose gel electrophoresis.CsgA is about 530bp.Bands are visible between 500bp and 750bp.The deficiency of band of knock-out strain is as expected.
SDS-PAGE results:
To verify the over-expression of curli, we did Cango Red(CR) deletion assay.The CR can binds to amyloid proteins and thus can be pulled down to the pellet while the absorbance of supernatant will reduced.The results are as below.
Quantitative Congo Red (CR) Binding Assays to verify the successful expression of Curli. (A) a is a wild-type E.coli strain that normally expresses csgA protein. b is the PHL628-△csgA cells (csgA knock out) strain. It can be seen that there is almost no expression of csgA after knocking out. (B) a is the wild type strain, b is the csgA overexpressing strain (a plasmid carrying a csga protein is expressed in wild type E.coli), and c is the csga KO cell. By adding Congo red dye to the bacteria mixture, centrifugation and shaking, we can compare the expression level of curli by observing the color depth of the precipitate. (C) Figure C is an inverted view of the EP tube in Figure B, showing the color of the sediment at the bottom of the centrifuge tube. a – WT. b – csgA KO, c – csgA overexpressing.
Quantitative results:
We measured the absorbance of supernatant at 480nm for CR and 600nm for cell concentration.The comparison of CgsA knockout Defect bacteria, wild type BL21(DE3) our recombinant csgA-expressing bacteria shows the overexpression of our recombinant strain.