Difference between revisions of "Part:BBa K4376002"

 
 
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It can be recognized by RNA polymerase and starts to transcribe as well as control the time and level of gene expression. In our project, tac promoter controls the expression of bpsA gene.
 
It can be recognized by RNA polymerase and starts to transcribe as well as control the time and level of gene expression. In our project, tac promoter controls the expression of bpsA gene.
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==Nanjing-China 2023 Characterization==
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===<strong>Expression of indigo in Corynebacterium glutamicum</strong>===
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We have successfully expressed bpsA in Corynebacterium glutamicum. As shown below, the right conical flask shows the fermentation results after introducing empty PEKEX2 into the C.glutamicum, whereas the left conical flask shows the fermentation results of indigoidine production after introducing bpsA plasmid into C.glutamicum. Obviously, the left one expresses bpsA successfully with fully blue in the fermentation broth.
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Below is a diagram of Thomas Brilliant Blue staining of Corynebacterium glutamicum. From left to right, the first lane is the whole cell lysate of C. glutamicum, the second lane is the whole cell lysate after introduction of the plasmid, the third lane is the supernatant of wild-type C. glutamicum, and the fourth lane is the supernatant after introduction of the plasmid. It indicates that bpsA successfully expressed indigo after introduction of the plasmid.
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<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/protein.png" width="500" height="400"/>
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===<strong>Direct Dyeing</strong>===
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We stained the bacterial cellulose membranes directly with C. glutamicum cultures.
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This is an electron microscope image after direct staining. Microfibers intertwine with each other to form a mesh-like structure, in which the indigoidine-secreting C. glutamicum are encapsulated.
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<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/1-i154.jpg" width="500" height="400"/>
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<html><style>
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img{margin:auto;}
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#a1{width:500px;height:400px;margin:auto;}
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</style><div id="a1">
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<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/1-i156.jpg" width="500" height="400"/>
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</div></html>
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===<strong>Co-culturing</strong>===
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In order to lay the groundwork for the subsequent one-step production of colored fibers by expressing bpsA directly in K.xylinus, we first started with a co-culture of K. xylinus and C. glutamicum as a way to further explore the way indigoidine binds to bacterial cellulose as well as the physical and chemical properties. The reason we choose K.xylinus is because it is reported as one of the high cellulose-producing strains by journal articles. Unfortunately, we were not able to obtain colored BC membranes first, but rather colored granular bacterial cellulose. In subsequent experiments, we choose the static culture conditions and utilize BC membranes as a framework to grow C. glutamicum. This novel idea offers us a paradigm to obtain the colored BC membranes with different patterns determined by how we inoculate C. glutamicum.
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<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/co-culture-cartoon.png" width="500" height="400"/>
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<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/cocultivate-1.jpg" width="500" height="500"/>
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Latest revision as of 12:29, 11 October 2023


control the expression of bpsA gene

It can be recognized by RNA polymerase and starts to transcribe as well as control the time and level of gene expression. In our project, tac promoter controls the expression of bpsA gene.

Nanjing-China 2023 Characterization

Expression of indigo in Corynebacterium glutamicum

We have successfully expressed bpsA in Corynebacterium glutamicum. As shown below, the right conical flask shows the fermentation results after introducing empty PEKEX2 into the C.glutamicum, whereas the left conical flask shows the fermentation results of indigoidine production after introducing bpsA plasmid into C.glutamicum. Obviously, the left one expresses bpsA successfully with fully blue in the fermentation broth.


Below is a diagram of Thomas Brilliant Blue staining of Corynebacterium glutamicum. From left to right, the first lane is the whole cell lysate of C. glutamicum, the second lane is the whole cell lysate after introduction of the plasmid, the third lane is the supernatant of wild-type C. glutamicum, and the fourth lane is the supernatant after introduction of the plasmid. It indicates that bpsA successfully expressed indigo after introduction of the plasmid.

Direct Dyeing

We stained the bacterial cellulose membranes directly with C. glutamicum cultures.


This is an electron microscope image after direct staining. Microfibers intertwine with each other to form a mesh-like structure, in which the indigoidine-secreting C. glutamicum are encapsulated.


Co-culturing

In order to lay the groundwork for the subsequent one-step production of colored fibers by expressing bpsA directly in K.xylinus, we first started with a co-culture of K. xylinus and C. glutamicum as a way to further explore the way indigoidine binds to bacterial cellulose as well as the physical and chemical properties. The reason we choose K.xylinus is because it is reported as one of the high cellulose-producing strains by journal articles. Unfortunately, we were not able to obtain colored BC membranes first, but rather colored granular bacterial cellulose. In subsequent experiments, we choose the static culture conditions and utilize BC membranes as a framework to grow C. glutamicum. This novel idea offers us a paradigm to obtain the colored BC membranes with different patterns determined by how we inoculate C. glutamicum.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]