Difference between revisions of "Part:BBa K4605010"
(→‎Expression of indigoidine in Corynebacterium glutamicum)
 
(10 intermediate revisions by 2 users not shown)
Line 1: Line 1:
  
 
__NOTOC__
 
__NOTOC__
−
<partinfo>BBa_K4605010 short</partinfo>
+
<partinfo>BBa_K4605002 short</partinfo>
  
−
aaaa
+
==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.
 +
 
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a1{width:350px;height:400px;margin:0px 0px 0px 8px;border:3px solid grey}
 +
 
 +
</style><div id="a1">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/new-notion.png" width="350" height="400"/>
 +
</div></html>
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a77{width:560px;height:400px;margin:-412px 200px 15px 375px;border:3px solid grey}
 +
</style><div id="a77">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/pekex2.png" width="560" height="400"/>
 +
</div></html>
 +
 
 +
==Experiment==
 +
===<strong>Expression of indigoidine in Corynebacterium glutamicum</strong>===
 +
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.
 +
<html><style>
 +
img{margin:auto;}
 +
#a2{width:500px;height:400px;margin:auto;border:3px solid grey}
 +
</style><div id="a2">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/k-and-glu2.jpg" width="500" height="400"/>
 +
</div></html>
 +
<p>
 +
At the same time, we used DMSO to directly suspend the bacteria for ultrasound, and then centrifuge to obtain the upper clearing to measure the absorption peak, and the absorption peak was about 590nm, which proved that it was indeed indigoidine.
 +
<html><style>
 +
img{margin:auto;}
 +
#a3{width:550px;height:400px;margin:auto;border:3px solid grey}
 +
</style><div id="a3">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/xishoufeng.jpg" width="550" height="400"/>
 +
</div></html>
 +
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a4{width:400px;height:400px;margin:20px 20px 20px 270px;border:3px solid grey}
 +
</style><div id="a4">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/indigoidine-600.jpg" width="400" height="400"/>
 +
</div></html>
 +
 
 +
 
 +
 
 +
 
 +
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.
 +
<html><style>
 +
img{margin:auto;}
 +
#a5{width:500px;height:400px;margin:auto;border:3px solid grey}
 +
</style><div id="a5">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/protein.png" width="500" height="400"/>
 +
</div></html>
 +
 
 +
===<strong>Direct Dyeing</strong>===
 +
We stained the bacterial cellulose membranes directly with C. glutamicum cultures.
 +
<html><style>
 +
img{margin:auto;}
 +
#a7{width:300px;height:400px;margin:15px 200px 15px 320px;border:3px solid grey}
 +
</style><div id="a7">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/direct-dye1.jpg" width="300" height="400"/>
 +
</div></html>
 +
 
 +
===<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.
 +
<html><style>
 +
img{margin:auto;}
 +
#a10{width:500px;height:400px;margin:auto;border:3px solid grey}
 +
</style><div id="a10">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/co-culture-cartoon.png" width="500" height="400"/>
 +
</div></html>
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a11{width:400px;height:400px;margin:auto;border:3px solid grey}
 +
</style><div id="a11">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/cocultivate-1.jpg" width="400" height="400"/>
 +
</div></html>
 +
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.
 +
<html><style>
 +
img{margin:auto;}
 +
#a8{width:400px;height:300px;margin:15px 200px 15px 60px;border:3px solid black}
 +
</style><div id="a8">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/1-i154.jpg" width="400" height="300"/>
 +
</div></html>
 +
 
 +
<html><style>
 +
img{margin:auto;}
 +
#a9{width:400px;height:300px;margin:-320px 20px 25px 470px;border:3px solid black}
 +
</style><div id="a9">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/1-i156.jpg" width="400" height="300"/>
 +
</div></html>
 +
 
 +
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.
 +
<html><style>
 +
img{margin:auto;}
 +
#a20{width:400px;height:300px;margin:20px 20px 25px 60px;border:3px solid grey}
 +
</style><div id="a20">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/wet-lab/coculture-bottle.png" width="400" height="300"/>
 +
</div></html>
 +
<html><style>
 +
img{margin:auto;}
 +
#a21{width:400px;height:300px;margin:-330px 20px 25px 470px;border:3px solid grey}
 +
</style><div id="a21">
 +
<img src="https://static.igem.wiki/teams/4605/wiki/gongpei.png" width="400" height="300"/>
 +
</div></html>
 +
 
 +
===<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
  
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
−
===Usage and Biology===
 
  
 
<!-- -->
 
<!-- -->
−
 
 
<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
−
https://static.igem.wiki/teams/4605/wiki/2000marker.png
 
 
<partinfo>BBa_K4605010 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K4605010 SequenceAndFeatures</partinfo>
  
  
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
−
===Functional Parameters===
+
===Functional Parameter===
 
<partinfo>BBa_K4605010 parameters</partinfo>
 
<partinfo>BBa_K4605010 parameters</partinfo>
 
<!-- -->
 
<!-- -->

Latest revision as of 12:28, 11 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.

At the same time, we used DMSO to directly suspend the bacteria for ultrasound, and then centrifuge to obtain the upper clearing 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.

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