Difference between revisions of "Part:BBa K2533017"
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<h1>'''Characterization'''</h1>
 
<h1>'''Characterization'''</h1>
 
We add FLAG-tag and His-tag to help us to detect whether the protein has been expressed or not.
 
We add FLAG-tag and His-tag to help us to detect whether the protein has been expressed or not.
−
[[File:T--HUST-China--2018-（ldhDC）-（1）.png‎|400px|thumb|center|Figure1:RBS-ldhD-FLAG-lldP-his tag]]
+
[[File:T--HUST-China--2018-（ldhDC）-（1）.png‎|400px|thumb|center|Figure1. RBS-ldhD-FLAG-lldP-his tag]]
  
 
<h2> Fluorescence image</h2>
 
<h2> Fluorescence image</h2>
 
To make sure that we get the target gene, we did the detection of fluorescence microscopy .And here is the result.Needless to see that the gene expressed well. The fluorescence of YFP is much more brighter than negative control.Therefore ,to some extant the expression is was successful.
 
To make sure that we get the target gene, we did the detection of fluorescence microscopy .And here is the result.Needless to see that the gene expressed well. The fluorescence of YFP is much more brighter than negative control.Therefore ,to some extant the expression is was successful.
−
[[File:T--HUST-China--2018-（YFP）-（1）.png|400px|thumb|center|Figure2: fluorescence microscopy]]
+
[[File:T--HUST-China--2018-（YFP）-（1）.png|400px|thumb|center|Figure2. fluorescence microscopy]]
  
 
<h2>SDS page</h2>
 
<h2>SDS page</h2>
−
[[File:T--HUST-China--2018-（page）-（1）.png|400px|thumb|center|Figure3:SDS page]]
+
[[File:T--HUST-China--2018-（page）-（1）.png|400px|thumb|center|Figure3. SDS page]]
 
There were nearly 0.2g bacteria used to testify the expression of lldP. After affinity chromatography, the protein was electrophoresed through SDS-PAGE. As the red box shows, lldP can be seen . Comparing to wild type, lldP is expressed in Synechocystis sp. PCC 6803.
 
There were nearly 0.2g bacteria used to testify the expression of lldP. After affinity chromatography, the protein was electrophoresed through SDS-PAGE. As the red box shows, lldP can be seen . Comparing to wild type, lldP is expressed in Synechocystis sp. PCC 6803.
  
 
<h2>lactate detect</h2>
 
<h2>lactate detect</h2>
−
[[File:T--HUST-China--2018-（乳酸）-（1）.png|400px|thumb|center|Figure3:Result of lactate detecting]]
+
[[File:T--HUST-China--2018-（乳酸）-（1）.png|400px|thumb|center|Figure3. Result of lactate detecting]]
 
Lactate production of engineered Synechocystis PCC6803. Comparison of WT, ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP. Synechocystis grew for 7 days(OD=1), then L-Rhamose(1g/L) was added. After induced 72 hours, lactate concentration in culture supernatant has shown above.Obviously ,we can see the engineer sphaerocystis produced more lactate.
 
Lactate production of engineered Synechocystis PCC6803. Comparison of WT, ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP. Synechocystis grew for 7 days(OD=1), then L-Rhamose(1g/L) was added. After induced 72 hours, lactate concentration in culture supernatant has shown above.Obviously ,we can see the engineer sphaerocystis produced more lactate.
  

Revision as of 22:45, 17 October 2018


RBS-ldhDC-FLAG-lldP-his tag

Show the expression of the protein.


Characterization

We add FLAG-tag and His-tag to help us to detect whether the protein has been expressed or not.

Figure1. RBS-ldhD-FLAG-lldP-his tag

Fluorescence image

To make sure that we get the target gene, we did the detection of fluorescence microscopy .And here is the result.Needless to see that the gene expressed well. The fluorescence of YFP is much more brighter than negative control.Therefore ,to some extant the expression is was successful.

Figure2. fluorescence microscopy

SDS page

Figure3. SDS page

There were nearly 0.2g bacteria used to testify the expression of lldP. After affinity chromatography, the protein was electrophoresed through SDS-PAGE. As the red box shows, lldP can be seen . Comparing to wild type, lldP is expressed in Synechocystis sp. PCC 6803.

lactate detect

Figure3. Result of lactate detecting

Lactate production of engineered Synechocystis PCC6803. Comparison of WT, ldhD-lldP, ldhDc-lldP, ldhDnARSdR-lldP, ldhDARSdR-lldP. Synechocystis grew for 7 days(OD=1), then L-Rhamose(1g/L) was added. After induced 72 hours, lactate concentration in culture supernatant has shown above.Obviously ,we can see the engineer sphaerocystis produced more lactate.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 780
    Illegal BglII site found at 1357
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1493
    Illegal AgeI site found at 1956
    Illegal AgeI site found at 2394
    Illegal AgeI site found at 2553
    Illegal AgeI site found at 2565
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1067