Difference between revisions of "Part:BBa K2533038"
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<h1>'''Usage and biology'''</h1> | <h1>'''Usage and biology'''</h1> | ||
| − | pflB helps to transform pyruvate into Acetyl-CoA and fdh helps to transform formate into | + | pflB helps to transform pyruvate into Acetyl-CoA and fdh helps to transform formate into CO<sub>2</sub>. With the overexpression of pflB-fdh, Shewanella could produce NADH more efficiently, which brings more electricity to be produced. |
<h1>'''Characterization'''</h1> | <h1>'''Characterization'''</h1> | ||
This is one section contained two genes for NADH production part. | This is one section contained two genes for NADH production part. | ||
| − | [[File:T--HUST-China--2018-tonglu-pflB-fdh.png |400px|thumb|center|Figure1 | + | [[File:T--HUST-China--2018-tonglu-pflB-fdh.png |400px|thumb|center|Figure1. RBS-pflB-RBS-fdh]] |
<h2>DNA Gel Electrophoretic</h2> | <h2>DNA Gel Electrophoretic</h2> | ||
To make sure that we get the target gene, we did the DNA gel electrophoretic to separate different gene. And here is the result. | To make sure that we get the target gene, we did the DNA gel electrophoretic to separate different gene. And here is the result. | ||
| − | [[File:T--HUST-China--2018-Notebook-gel13.jpeg|400px|thumb|center|Figure2 | + | [[File:T--HUST-China--2018-Notebook-gel13.jpeg|400px|thumb|center|Figure2. Verification of successful transformation of pYYDT-pflB-fdh]] |
| − | Our target length is 9314bp, and as the marker is marker4, we could be sure that the bright | + | Our target length is 9314bp, and as the marker is marker4, we could be sure that the bright band in this picture is our target gene. |
<h2>Real-Time Quantitative PCR</h2> | <h2>Real-Time Quantitative PCR</h2> | ||
To demonstrate that pflB-fdh could be overexpressed by engineered Shewanella, we did Real-Time Quantitative PCR. | To demonstrate that pflB-fdh could be overexpressed by engineered Shewanella, we did Real-Time Quantitative PCR. | ||
| − | [[File:T--HUST–China--2018-result-pflB-fdh.jpeg |400px|thumb|center|Figure3 | + | [[File:T--HUST–China--2018-result-pflB-fdh.jpeg |400px|thumb|center|Figure3. Relative expression level of pflB-fdh in engineered Shewanella Oneidensis MR-1.]] |
There was no signal in bacteria which contained pYYDT so we chose pYYDT-pflB-fdh as standard quantity. | There was no signal in bacteria which contained pYYDT so we chose pYYDT-pflB-fdh as standard quantity. | ||
<h2>Electrogenesis</h2> | <h2>Electrogenesis</h2> | ||
By comparing the ability of producing electricity, we might find out whether pflB-fdh could effectively help Shewanella to produce more electricity. | By comparing the ability of producing electricity, we might find out whether pflB-fdh could effectively help Shewanella to produce more electricity. | ||
| − | [[File:T--HUST-China--2018-elec-gapA-mdh.png |400px|thumb|center|Figure4 | + | [[File:T--HUST-China--2018-elec-gapA-mdh.png |400px|thumb|center|Figure4. The comparison of electricity production between Shewanella contained pYYDT and pYYDT-pflB-fdh.]] |
It could be demonstrated that targeted gene could be expressed in the engineered cells. More NADH has been produced by engineered bacteria, which helps to produce more electricty. | It could be demonstrated that targeted gene could be expressed in the engineered cells. More NADH has been produced by engineered bacteria, which helps to produce more electricty. | ||
Latest revision as of 22:57, 17 October 2018
RBS-pflB-RBS-fdh
It encodes pyruvate formate-lyase and formate dehydrogenase cytochrome b.
Usage and biology
pflB helps to transform pyruvate into Acetyl-CoA and fdh helps to transform formate into CO2. With the overexpression of pflB-fdh, Shewanella could produce NADH more efficiently, which brings more electricity to be produced.
Characterization
This is one section contained two genes for NADH production part.
DNA Gel Electrophoretic
To make sure that we get the target gene, we did the DNA gel electrophoretic to separate different gene. And here is the result.
Our target length is 9314bp, and as the marker is marker4, we could be sure that the bright band in this picture is our target gene.
Real-Time Quantitative PCR
To demonstrate that pflB-fdh could be overexpressed by engineered Shewanella, we did Real-Time Quantitative PCR.
There was no signal in bacteria which contained pYYDT so we chose pYYDT-pflB-fdh as standard quantity.
Electrogenesis
By comparing the ability of producing electricity, we might find out whether pflB-fdh could effectively help Shewanella to produce more electricity.
It could be demonstrated that targeted gene could be expressed in the engineered cells. More NADH has been produced by engineered bacteria, which helps to produce more electricty.