Difference between revisions of "Part:BBa K2533045"
 
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<partinfo>BBa_K2533045 short</partinfo>
 
<partinfo>BBa_K2533045 short</partinfo>
  
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produce latic acid
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produce lactate
  
 
<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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mleS refers to malate dehydrogenase, performing the conversion of malic acid to L-lactate. Rhodopseudomonas palustris could produce lactate more efficiently, which brings Shewanella more carbon source.
  
 
<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 lactate producing part.
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[[File:T--HUST-China--2018-tonglu-mleS.png ‎|400px|thumb|center|Figure1:RBS-mles]]
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[[File:T--HUST-China--2018-tonglu-mleS.png ‎|400px|thumb|center|Figure1. RBS-mles]]
  
 
<h2>DNA Gel Electrophoretic</h2>
 
<h2>DNA Gel Electrophoretic</h2>
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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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To make sure that we get the target gene, we did DNA gel electrophoretic for verification. And here is the result.
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[[File:T--HUST-China--2018-tonglu-mles.png|400px|thumb|center|Figure2:Verification of successful transformation of pSB1C3-RBS-mleS]]
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[[File:T--HUST-China--2018-tonglu-mles.png|400px|thumb|center|Figure2. Verification of successful transformation of pSB1C3-RBS-mleS]]
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Our target genes are 1969bp, 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 1641bp, 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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<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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[[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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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.
+
  
 
<h2>Electrogenesis</h2>
 
<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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By detecting the production of lactate after expressing, we might find out whether mleS could effectively help Rhodopseudomonas palustris produce more lactate.
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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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[[File:T--HUST-China--2018-expression of lactate.png ‎|400px|thumb|center|Figure3. shows that our modification is effective. Every gene circuits can help strains produce lactate, and mleS-lldP-ldhA is the most efficient one. Therefore, our construction of gene circuits achieves the goal to help strains produce lactate.]]
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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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It could be demonstrated that the target genes could be expressed in the engineered cells. More lactate has been produced by engineered bacteria.  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  

Latest revision as of 23:04, 17 October 2018


RBS-mleS

produce lactate

Usage and biology

mleS refers to malate dehydrogenase, performing the conversion of malic acid to L-lactate. Rhodopseudomonas palustris could produce lactate more efficiently, which brings Shewanella more carbon source.

Characterization

This is one section for lactate producing part.

Figure1. RBS-mles

DNA Gel Electrophoretic

To make sure that we get the target gene, we did DNA gel electrophoretic for verification. And here is the result.

Figure2. Verification of successful transformation of pSB1C3-RBS-mleS

Our target gene is 1641bp, and as the marker is DS5000, we could be sure that the bright bands in this picture are our target genes.

Electrogenesis

By detecting the production of lactate after expressing, we might find out whether mleS could effectively help Rhodopseudomonas palustris produce more lactate.

Figure3. shows that our modification is effective. Every gene circuits can help strains produce lactate, and mleS-lldP-ldhA is the most efficient one. Therefore, our construction of gene circuits achieves the goal to help strains produce lactate.

It could be demonstrated that the target genes could be expressed in the engineered cells. More lactate has been produced by engineered bacteria.