Difference between revisions of "Part:BBa K4376001"
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We amplified two bpsA fragments then transformed recombinant vector into DH5α E.coli strain and Corynebacterium glutamicum.
 
We amplified two bpsA fragments then transformed recombinant vector into DH5α E.coli strain and Corynebacterium glutamicum.
 
[[File:engineering-1.png|600px||centre]]
 
[[File:engineering-1.png|600px||centre]]
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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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===<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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===<strong>Expression of bpsA in K. xylinus</strong>===
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Because K. xylinus does not have the native PPTase that is necessary for activating apo-form of indigoidine synthase into its active holo-form by adding coenzyme A to the peptide carrier domain (PCP), we need to transfect the target gene both bpsA and pcpS (encoding PPTase)into K. xylinus using pSB1A2 as a plasmid vector, and synthesize indigoidine fibers using K. xylinus which is capable of producing cellulose in high yield.With previous basic explorations, we will use PSB1A2 plasmid backbone, ligated with promoters such as strong promoters (J23104,J23100,J23109 etc.), and CDS sequences to express bpsA and pcpS in K. xylinus while binding to bacterial cellulose membranes.
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<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here

Revision as of 05:58, 8 October 2023


Produce indigoidine

Its encoding protein is blue-pigment indigoidine synthetase, which carries strong fluxes toward L-glutamate, a precursor of indigoidine. In our project, we sue it to produce indigoidine.

We amplified two bpsA fragments then transformed recombinant vector into DH5α E.coli strain and Corynebacterium glutamicum.

Engineering-1.png


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.






Expression of bpsA in K. xylinus

Because K. xylinus does not have the native PPTase that is necessary for activating apo-form of indigoidine synthase into its active holo-form by adding coenzyme A to the peptide carrier domain (PCP), we need to transfect the target gene both bpsA and pcpS (encoding PPTase)into K. xylinus using pSB1A2 as a plasmid vector, and synthesize indigoidine fibers using K. xylinus which is capable of producing cellulose in high yield.With previous basic explorations, we will use PSB1A2 plasmid backbone, ligated with promoters such as strong promoters (J23104,J23100,J23109 etc.), and CDS sequences to express bpsA and pcpS in K. xylinus while binding to bacterial cellulose membranes.


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 3887
    Illegal XhoI site found at 3040
    Illegal XhoI site found at 3739
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 286
    Illegal NgoMIV site found at 693
    Illegal AgeI site found at 1951
    Illegal AgeI site found at 2004
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 229
    Illegal BsaI site found at 1540
    Illegal BsaI site found at 2254
    Illegal BsaI.rc site found at 358
    Illegal BsaI.rc site found at 3544
    Illegal SapI.rc site found at 1792