Difference between revisions of "Part:BBa K4605010"

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==Description==
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BpsA stands for the Blue-pigment indigoidine synthetase gene.Itself is derived from Streptomyces lavendulae and is used in the synthesis of indigo. It can only be activated from inative apo-form to the active holo-bpsA by the addition of CoA to its PCP, catalyzed by PPTase, which synthesizes two molecules of glutamine into one molecule of indigo. Corynebacterium glutamicum is usually used to express bpsA for high indigo production.
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In this experiment we will modify Komagataeibacter xylinus to express bpsA for one-step synthesis of colored fibers, and also codon optimize the bpsA coding sequence.
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<img src="https://static.igem.wiki/teams/4605/wiki/principle-002.png" width="350" height="400"/>
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==Experiment==
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===<strong>Expression of indigo in Corynebacterium glutamicum</strong>===
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We 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 indigo production after introducing bpsA plasmid into C.glutamicum.
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<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/k-and-glu2.jpg" width="500" height="400"/>
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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 Valley Stick, 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{margin:auto;}
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<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/protein-edit.png" width="300" height="400"/>
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Prediction of alpha fold of BpsA-expressed proteins
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===<strong>Direct Dyeing</strong>===
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We stained the bacterial cellulose membranes directly with indigo-containing grain stick cultures
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<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/direct-dye1.jpg" width="300" height="400"/>
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This is an electron microscope image after direct staining
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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 pave the way 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 with C. glutamicum as a way to further explore the way indigo binds to bacterial cellulose as well as the physical and chemical properties. Unfortunately, we were not able to obtain colored membrane BC first, but rather colored granular bacterial cellulose.
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img{margin:auto;}
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<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/cocultivate-1.jpg" width="400" height="400"/>
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</div></html>
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===<strong>Expression of bpsA in K. xylinus</strong>===
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With previous basic explorations, we will use a wood vinegar compatible PSB1A2 plasmid backbone, ligated with promoters such as strong promoters (J23104, J23102, etc.), and bpsA sequences to try to express bpsA in K. xylinus while binding to bacterial cellulose membranes.
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===<strong>References</strong>===
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[1] Mohammad Rifqi Ghiffary, Cindy Pricilia Surya Prabowo, Komal Sharma, Yuchun Yan, Sang Yup Lee, and Hyun Uk Kim.High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622
  
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4605010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4605010 SequenceAndFeatures</partinfo>
  

Revision as of 12:35, 17 September 2023


Used to produce indigo in Corynebacterium glutamicum

Description

BpsA stands for the Blue-pigment indigoidine synthetase gene.Itself is derived from Streptomyces lavendulae and is used in the synthesis of indigo. It can only be activated from inative apo-form to the active holo-bpsA by the addition of CoA to its PCP, catalyzed by PPTase, which synthesizes two molecules of glutamine into one molecule of indigo. Corynebacterium glutamicum is usually used to express bpsA for high indigo production.

In this experiment we will modify Komagataeibacter xylinus to express bpsA for one-step synthesis of colored fibers, and also codon optimize the bpsA coding sequence.

Experiment

Expression of indigo in Corynebacterium glutamicum

We 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 indigo production after introducing bpsA plasmid into C.glutamicum.

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 Valley Stick, 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.

Prediction of alpha fold of BpsA-expressed proteins

Direct Dyeing

We stained the bacterial cellulose membranes directly with indigo-containing grain stick cultures

This is an electron microscope image after direct staining

Co-culturing

In order to pave the way 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 with C. glutamicum as a way to further explore the way indigo binds to bacterial cellulose as well as the physical and chemical properties. Unfortunately, we were not able to obtain colored membrane BC first, but rather colored granular bacterial cellulose.

Expression of bpsA in K. xylinus

With previous basic explorations, we will use a wood vinegar compatible PSB1A2 plasmid backbone, ligated with promoters such as strong promoters (J23104, J23102, etc.), and bpsA sequences to try to express bpsA in K. xylinus while binding to bacterial cellulose membranes.

References

[1] Mohammad Rifqi Ghiffary, Cindy Pricilia Surya Prabowo, Komal Sharma, Yuchun Yan, Sang Yup Lee, and Hyun Uk Kim.High-Level Production of the Natural Blue Pigment Indigoidine from Metabolically Engineered Corynebacterium glutamicum for Sustainable Fabric Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 3789
    Illegal EcoRI site found at 3940
    Illegal XbaI site found at 25
    Illegal XbaI site found at 9532
    Illegal SpeI site found at 7594
    Illegal SpeI site found at 7973
    Illegal PstI site found at 13
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 3789
    Illegal EcoRI site found at 3940
    Illegal SpeI site found at 7594
    Illegal SpeI site found at 7973
    Illegal PstI site found at 13
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 3789
    Illegal EcoRI site found at 3940
    Illegal BglII site found at 3932
    Illegal BamHI site found at 31
    Illegal XhoI site found at 3085
    Illegal XhoI site found at 3784
    Illegal XhoI site found at 6063
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 3789
    Illegal EcoRI site found at 3940
    Illegal XbaI site found at 25
    Illegal XbaI site found at 9532
    Illegal SpeI site found at 7594
    Illegal SpeI site found at 7973
    Illegal PstI site found at 13
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 3789
    Illegal EcoRI site found at 3940
    Illegal XbaI site found at 25
    Illegal XbaI site found at 9532
    Illegal SpeI site found at 7594
    Illegal SpeI site found at 7973
    Illegal PstI site found at 13
    Illegal NgoMIV site found at 331
    Illegal NgoMIV site found at 738
    Illegal NgoMIV site found at 9257
    Illegal NgoMIV site found at 10601
    Illegal AgeI site found at 1996
    Illegal AgeI site found at 2049
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 274
    Illegal BsaI site found at 1585
    Illegal BsaI site found at 2299
    Illegal BsaI site found at 4139
    Illegal BsaI.rc site found at 403
    Illegal BsaI.rc site found at 3589
    Illegal SapI site found at 5029
    Illegal SapI.rc site found at 1837