Difference between revisions of "Part:BBa K2533035"
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<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- | + | [[File:T--HUST-China--2018-tonglu-gapA-mdh.png |400px|thumb|center|Figure1. RBS-gapA-RBS-mdh]] |
<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- | + | [[File:T--HUST-China--2018-Notebook-gel11.jpeg|400px|thumb|center|Figure2. Verification of successful transformation of pYYDT-gapA-mdh]] |
| − | Our target | + | Our target gene is 2666bp, and as the marker is DS5000, 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 gapA could be overexpressed by engineered Shewanella, we did Real-Time Quantitative PCR. | + | To demonstrate that gapA-mdh could be overexpressed by engineered Shewanella, we did Real-Time Quantitative PCR. |
| − | [[File:T--HUST–China--2018-result-gapA-mdh.jpeg |400px|thumb|center|Figure3 | + | [[File:T--HUST–China--2018-result-gapA-mdh.jpeg |400px|thumb|center|Figure3. Relative expression level of gapA-mdh in engineered Shewanella Oneidensis MR-1.]] |
| − | There was no signal in bacteria which contained pYYDT so we chose pYYDT-gapA as standard quantity. | + | There was no signal in bacteria which contained pYYDT so we chose pYYDT-gapA-mdh as standard quantity. |
<h2>Electrogenesis</h2> | <h2>Electrogenesis</h2> | ||
| − | By comparing the ability of producing electricity, we might find out whether | + | By comparing the ability of producing electricity, we might find out whether gapA-mdh 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-gapA-mdh.]] |
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 22:55, 17 October 2018
RBS-gapA-RBS-mdh-TT
It encodes glyceraldehyde-3-phosphate dehydrogenase and NAD dependent malate dehydrogenase.
Usage and biology
gapA could transform 3- phosphoglyceraldehyde into 1,3- diphosphoglycerate and mdh could transform malate into pyruvate. With the overexpression of gapA-mdh, 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 gene is 2666bp, and as the marker is DS5000, we could be sure that the bright band in this picture is our target gene.
Real-Time Quantitative PCR
To demonstrate that gapA-mdh 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-gapA-mdh as standard quantity.
Electrogenesis
By comparing the ability of producing electricity, we might find out whether gapA-mdh 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.