Difference between revisions of "Part:BBa K4605002"
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==Description==
 
==Description==
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BpsA stands for the blue-pigment indigoidine synthetase gene,encoded by a single module-type nonribosomal peptide synthetase (NRPS).It can synthesize two molecules of glutamine into one molecule of indigo.Itself is derived from Streptomyces lavendulae.
+
BpsA stands for the blue pigment indigoidine synthetase gene, encoding a single module type non-ribosomal peptide synthetase called BpsA. Indigoidine synthetase can synthesize two molecules of glutamine into one molecule of indigoidine. Itself is derived from Streptomyces lavendulae.
  
−
But Corynebacterium glutamicum is usually used to express bpsA for high indigo production,because it carries strong fluxes for the biosynthesis of L-glutamate, a precursor of L-glutamine.Meanwhile,C. glutamate can also heterologously express indigo synthase and has the pcpS gene,which expresses PPTase(4'-phosphopantetheinyl transferase).The PPTase is of great significance becasuse it mediates the activation of the apo-form of BpsA into its active holo-form by adding coenzyme A to the peptide carrier domain (PCP).
+
Corynebacterium glutamicum is the ideal host for the expression of bpsA to achieve high indigoidine production, because it carries strong fluxes of L-glutamate, a precursor of L-glutamine and L-glutamine is the substrate of the indioigdine synthetase. Meanwhile, C. glutamicum also has the native pcpS gene, which expresses PPTase(4'-phosphopantetheinyl transferase). The PPTase is of great significance because it converts the apo-form of the BpsA into its active holo-form by attaching coenzyme A to the peptide carrier domain (PCP).
 +
 
 +
In this project first we will obtain indigoidine, the chemical structure of which is 5,5-diamino-4,4-dihydroxy-3,3-diazadiphenoquinone-(2,2), by introducing pEKEX2 plasmid backbone ligated with bpsA, into C. glutamicum. In the next step, we would genetically modify Komagataeibacter xylinus and introduce PSB1A2 plasmid backbone ligated with bpsA and pcpS for one-step synthesis of colored fibers, and also codon optimize the bpsA and pcpS coding sequences to meet our needs.
  
−
In this experiment we will optimize Komagataeibacter xylinus to express bpsA for one-step synthesis of colored fibers, and also codon optimize the bpsA coding sequence.
 
  
 
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==Experiment==
 
==Experiment==
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===<strong>Expression of indigo in Corynebacterium glutamicum</strong>===
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===<strong>Expression of indigoidine in Corynebacterium glutamicum</strong>===
−
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.
+
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 K.xylinus, 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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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 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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Prediction of alpha fold of BpsA-expressed proteins
 
  
 
===<strong>Direct Dyeing</strong>===
 
===<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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We stained the bacterial cellulose membranes directly with C. glutamicum 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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 +
===<strong>Co-culturing</strong>===
 +
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.
 
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===<strong>Co-culturing</strong>===
+
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.
−
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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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>===
 
===<strong>Expression of bpsA in K. xylinus</strong>===
−
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.
+
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,J23119 etc.), and CDS sequences to express bpsA and pcpS in K. xylinus while binding to bacterial cellulose membranes.
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===<strong>References</strong>===
 
===<strong>References</strong>===
−
[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  
+
[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).
  
  

Latest revision as of 11:20, 10 October 2023


Blue-pigment indigoidine synthetase gene from Streptomyces lavendulae

Description

BpsA stands for the blue pigment indigoidine synthetase gene, encoding a single module type non-ribosomal peptide synthetase called BpsA. Indigoidine synthetase can synthesize two molecules of glutamine into one molecule of indigoidine. Itself is derived from Streptomyces lavendulae.

Corynebacterium glutamicum is the ideal host for the expression of bpsA to achieve high indigoidine production, because it carries strong fluxes of L-glutamate, a precursor of L-glutamine and L-glutamine is the substrate of the indioigdine synthetase. Meanwhile, C. glutamicum also has the native pcpS gene, which expresses PPTase(4'-phosphopantetheinyl transferase). The PPTase is of great significance because it converts the apo-form of the BpsA into its active holo-form by attaching coenzyme A to the peptide carrier domain (PCP).

In this project first we will obtain indigoidine, the chemical structure of which is 5,5-diamino-4,4-dihydroxy-3,3-diazadiphenoquinone-(2,2), by introducing pEKEX2 plasmid backbone ligated with bpsA, into C. glutamicum. In the next step, we would genetically modify Komagataeibacter xylinus and introduce PSB1A2 plasmid backbone ligated with bpsA and pcpS for one-step synthesis of colored fibers, and also codon optimize the bpsA and pcpS coding sequences to meet our needs.


Experiment

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 K.xylinus, 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.

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.

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.

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,J23119 etc.), and CDS sequences to express bpsA and pcpS 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

[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
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]