Difference between revisions of "Part:BBa K4605003"
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==Description==
 
==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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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.
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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).
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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.
  
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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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==Experiment==
 
==Experiment==
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===<strong>Expression of indigo in Corynebacterium glutamicum</strong>===
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===<strong>Promoters</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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We chose to use the Anderson family promoter, first, because it is compatible with K.xylinus, and there are enough references reporting that these promoters have been tested in several of bacteria such as G. xylinus 700178, G. hansenii 53582, and K. rhaeticus iGEM. Then we selected three of the strong promoters:J23100,J23104 and J23119.Here we choose J23104 promoter to test its performance. We linked it to bpsA-pcpS in the hope that colorful cellulose could be expressed.
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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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===<strong>Expression of bpsA in K. xylinus</strong>===
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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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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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This is an electron microscope image after direct staining
 
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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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===<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.
 
  
 
===<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 Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622  
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[2]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.
  
 
<!-- Add more about the biology of this part here
 
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Latest revision as of 10:53, 10 October 2023


Using strong promoters to improve the bpsA expression

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

Promoters

We chose to use the Anderson family promoter, first, because it is compatible with K.xylinus, and there are enough references reporting that these promoters have been tested in several of bacteria such as G. xylinus 700178, G. hansenii 53582, and K. rhaeticus iGEM. Then we selected three of the strong promoters:J23100,J23104 and J23119.Here we choose J23104 promoter to test its performance. We linked it to bpsA-pcpS in the hope that colorful cellulose could be expressed.

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 Dyes ACS Sustainable Chemistry & Engineering 2021 9 (19), 6613-6622

[2]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.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]