Difference between revisions of "Part:BBa K4605002"
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<partinfo>BBa_K4605002 short</partinfo>
 
<partinfo>BBa_K4605002 short</partinfo>
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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. 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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==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 cereal rods.
  
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Below is a diagram of Thomas Brilliant Blue staining of Corynebacterium glutamicum
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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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Prediction of alpha fold of BpsA-expressed proteins
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4605002 SequenceAndFeatures</partinfo>
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===<strong>Direct Staining BC</strong>===
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We stained the bacterial cellulose membranes directly with indigo-containing grain stick cultures
  
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===<strong>co-culturing</strong>===
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===Functional Parameter===
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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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<partinfo>BBa_K4605002 parameters</partinfo>
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==Design Page==
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===<strong>Expression of bpsA in wood K. xylinus</strong>===
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As you work on your project you will find that you've spent a large amount of time on the design of your parts and devices. As you approach your project with a Design->Build->Test cycle, it will be important to document the design considerations you've taken and the reasons for them. On the part's Design Page, detail any design considerations you may have taken for the part. This includes mutations to remove restriction sites, codon optimization, etc.
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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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<img src="https://static.igem.wiki/teams/4605/wiki/principle-002.png" width="210" height="180"  />
 
<img src="https://static.igem.wiki/teams/4605/wiki/principle-002.png" width="210" height="180"  />
 
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</html>
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<!-- Add more about the biology of this part here
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<!-- -->
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K4605002 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameter===
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<partinfo>BBa_K4605002 parameters</partinfo>
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Revision as of 07:40, 17 September 2023


Blue-pigment indigoidine synthetase gene from Streptomyces lavendulae

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 cereal rods.

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 Staining BC

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

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 wood 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.




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]