Difference between revisions of "Part:BBa K2533056"
 
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__NOTOC__
 
__NOTOC__
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<partinfo>BBa_K2533030 short</partinfo>
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<partinfo>BBa_K2533056 short</partinfo>
  
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It encodes D-lactate dehydrogenase.
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It encodes glyceraldehyde-3-phosphate dehydrogenase.
  
 
<h1>'''Usage and biology'''</h1>
 
<h1>'''Usage and biology'''</h1>
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dld refers to FAD-dependent D-lactate dehydrogenase which could catalyze D-lactate’s transformation into pyruvate. With the overexpression of dld, Shewanella could utilize D-lactate more efficiently, which brings more electricity being produced.
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It encodes glyceraldehyde-3-phosphate dehydrogenase which could transform 3- phosphoglyceraldehyde into 1,3- diphosphoglycerate. With the overexpression of gapA, Shewanella could produce NADH more efficiently, which brings more electricity to be produced.
  
 
<h1>'''Characterization'''</h1>
 
<h1>'''Characterization'''</h1>
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This is one section for lactate utilization part.
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This is one section for NADH production part.
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[[File:T--HUST-China--2018-tonglu-dld.png ‎|400px|thumb|center|Figure1:RBS-dld]]
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[[File:T--HUST-China--2018-tonglu-gapA.png ‎|400px|thumb|center|Figure1. RBS-gapA]]
  
 
<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.
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[[File:T--HUST-China--2018-Notebook-gel9.jpeg|400px|thumb|center|Figure2:Verification of successful transformation of pYYDT-dld]]
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[[File:T--HUST-China--2018-Notebook-gel3.jpeg|400px|thumb|center|Figure2. Verification of successful transformation of pYYDT-gapA]]
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Our target genes are 3366bp, and as the marker is DS5000, we could be sure that the bright bands in this picture are our target genes.
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Our target gene is 1126bp, 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>
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To demonstrate that dld could be overexpressed by engineered Shewanella, we did Real-Time Quantitative PCR.  
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To demonstrate that gapA could be overexpressed by engineered Shewanella, we did Real-Time Quantitative PCR.  
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[[File:T--HUST–China--2018-result-fig1.jpeg ‎|400px|thumb|center|Figure3:Relative expression level of dld in engineered Shewanella Oneidensis MR-1.]]
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[[File:T--HUST–China--2018-result-fig3.png ‎|400px|thumb|center|Figure3. Relative expression level of gapA in engineered Shewanella Oneidensis MR-1.]]
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As we can see from this figure, dld could be overexpressed by engineered Shewanella. For further verification, we used the engineered bacteria to produce electricity.
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There was no signal in bacteria which contained pYYDT so we chose pYYDT-gapA as standard quantity.
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<h2>Electrogenesis</h2>
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By comparing the ability of producing electricity, we might find out whether dld could effectively help Shewanella to produce more electricity.
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[[File:T--HUST-China--2018-elec-dld.png ‎|400px|thumb|center|Figure4:The comparison of electricity production between Shewanella contained pYYDT and pYYDT-dld.]]
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It could be demonstrated that targeted genes could be expressed in the engineered cells. More NADH has been produced by engineered bacteria, which helps to produce more electricty.  
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 22:28, 17 October 2018


PlacIq-lacI-Ptac-RBS-gapA

It encodes glyceraldehyde-3-phosphate dehydrogenase.

Usage and biology

It encodes glyceraldehyde-3-phosphate dehydrogenase which could transform 3- phosphoglyceraldehyde into 1,3- diphosphoglycerate. With the overexpression of gapA, Shewanella could produce NADH more efficiently, which brings more electricity to be produced.

Characterization

This is one section for NADH production part.

Figure1. RBS-gapA

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.

Figure2. Verification of successful transformation of pYYDT-gapA

Our target gene is 1126bp, 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 could be overexpressed by engineered Shewanella, we did Real-Time Quantitative PCR.

Figure3. Relative expression level of gapA in engineered Shewanella Oneidensis MR-1.

There was no signal in bacteria which contained pYYDT so we chose pYYDT-gapA as standard quantity.