Difference between revisions of "Part:BBa K4376003"

 
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Construction of the bpsA gene and tac inducible promoter to control the production of the blue-pigment indigoidine synthetase. The composite part is inserted into C.glutamicum in plasmid, resulting in the successful biosynthesis of indigodine pigment.
 
Construction of the bpsA gene and tac inducible promoter to control the production of the blue-pigment indigoidine synthetase. The composite part is inserted into C.glutamicum in plasmid, resulting in the successful biosynthesis of indigodine pigment.
  
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We amplified two bpsA fragments then transformed recombinant vector into DH5α E.coli strain and Corynebacterium glutamicum.
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[[File:engineering-1.png|600px||centre]]
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==Nanjing-China 2023's Characterization==
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===<strong>Expression of indigoidine 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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<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/k-and-glu2.jpg" width="500" height="400"/>
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We used DMSO to suspend C.glutamicum, and then sonicated the bacteria to break them apart. After centrifugation, we collected the supernatant to measure the absorption peak, and the absorption peak was about 590nm, which proved that it was indeed indigoidine.
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<img src="https://static.igem.wiki/teams/4605/wiki/xishoufeng.jpg" width="550" height="400"/>
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<img src="https://static.igem.wiki/teams/4605/wiki/indigoidine-600.jpg" width="400" height="400"/>
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Below is a diagram of SDS-PAGE 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 indigoidine 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 to test the binding capacity of microbial dye to bacterial cellulose membrane.
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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
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[2] Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622
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Fricke, P.M., Klemm, A., Bott, M. et al. On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases. Appl Microbiol Biotechnol 105, 3423–3456 (2021).
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[3] Goosens VJ, Walker KT, Aragon SM, Singh A, Senthivel VR, Dekker L, Caro-Astorga J, Buat MLA, Song W, Lee KY, Ellis T. Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria. ACS Synth Biol. 2021 Dec 17;10(12):3422-3434.
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[4]Florea M, Hagemann H, Santosa G, Abbott J, Micklem CN, Spencer-Milnes X, de Arroyo Garcia L, Paschou D, Lazenbatt C, Kong D, Chughtai H, Jensen K, Freemont PS, Kitney R, Reeve B, Ellis T. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proc Natl Acad Sci U S A. 2016 Jun 14;113(24):E3431-40.
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[5]Teh MY, Ooi KH, Danny Teo SX, Bin Mansoor ME, Shaun Lim WZ, Tan MH. An Expanded Synthetic Biology Toolkit for Gene Expression Control in Acetobacteraceae. ACS Synth Biol. 2019 Apr 19;8(4):708-723.
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[6]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 DyesACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622
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[7]Gilbert, C., Tang, TC., Ott, W. et al. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat. Mater. 20, 691–700 (2021).
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
 
===Usage and Biology===
 
===Usage and Biology===

Latest revision as of 15:18, 11 October 2023


the composite part is used to express the bpsA gene.

Construction of the bpsA gene and tac inducible promoter to control the production of the blue-pigment indigoidine synthetase. The composite part is inserted into C.glutamicum in plasmid, resulting in the successful biosynthesis of indigodine pigment.

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

Engineering-1.png

Nanjing-China 2023's Characterization

Expression of indigoidine 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.

We used DMSO to suspend C.glutamicum, and then sonicated the bacteria to break them apart. After centrifugation, we collected the supernatant to measure the absorption peak, and the absorption peak was about 590nm, which proved that it was indeed indigoidine.

Below is a diagram of SDS-PAGE 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 indigoidine after introduction of the plasmid.

Direct Dyeing

We stained the bacterial cellulose membranes directly with C. glutamicum cultures to test the binding capacity of microbial dye to bacterial cellulose membrane.

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

[2] Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622 Fricke, P.M., Klemm, A., Bott, M. et al. On the way toward regulatable expression systems in acetic acid bacteria: target gene expression and use cases. Appl Microbiol Biotechnol 105, 3423–3456 (2021).

[3] Goosens VJ, Walker KT, Aragon SM, Singh A, Senthivel VR, Dekker L, Caro-Astorga J, Buat MLA, Song W, Lee KY, Ellis T. Komagataeibacter Tool Kit (KTK): A Modular Cloning System for Multigene Constructs and Programmed Protein Secretion from Cellulose Producing Bacteria. ACS Synth Biol. 2021 Dec 17;10(12):3422-3434.

[4]Florea M, Hagemann H, Santosa G, Abbott J, Micklem CN, Spencer-Milnes X, de Arroyo Garcia L, Paschou D, Lazenbatt C, Kong D, Chughtai H, Jensen K, Freemont PS, Kitney R, Reeve B, Ellis T. Engineering control of bacterial cellulose production using a genetic toolkit and a new cellulose-producing strain. Proc Natl Acad Sci U S A. 2016 Jun 14;113(24):E3431-40.

[5]Teh MY, Ooi KH, Danny Teo SX, Bin Mansoor ME, Shaun Lim WZ, Tan MH. An Expanded Synthetic Biology Toolkit for Gene Expression Control in Acetobacteraceae. ACS Synth Biol. 2019 Apr 19;8(4):708-723.

[6]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 DyesACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622

[7]Gilbert, C., Tang, TC., Ott, W. et al. Living materials with programmable functionalities grown from engineered microbial co-cultures. Nat. Mater. 20, 691–700 (2021). 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 3893
    Illegal XhoI site found at 3046
    Illegal XhoI site found at 3745
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 292
    Illegal NgoMIV site found at 699
    Illegal AgeI site found at 1957
    Illegal AgeI site found at 2010
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 235
    Illegal BsaI site found at 1546
    Illegal BsaI site found at 2260
    Illegal BsaI.rc site found at 364
    Illegal BsaI.rc site found at 3550
    Illegal SapI.rc site found at 1798