Difference between revisions of "Part:BBa K4182000:Design"

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===Design Notes===
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==Profile==
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Name:VVD
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Base Pairs:453bp
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Origin:Neurospora crassa
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Performed E. coli codon optimization
 
Performed E. coli codon optimization
  
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==Usage&Biology==
  
  
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===Source===
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[[File:XJTU-1.png|300px|thumb|left|FIG. 1 Schematic diagram of blue light induction regulatory system]]
  
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Neurospora crassa，sequence found through NCBI(https://www.ncbi.nlm.nih.gov/gene/3873728)
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[[File:XJTU-2.png|300px|thumb|left|FIG. 2 Schematic diagram of blue light-induced regulation system]]
  
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===Research===
  
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===References===
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To realize the release of light-controlled products to achieve the goal of intelligent and sustained release, after an extensive literature survey, we found that the arabinose operon was modified to manipulate downstream gene expression, not through arabinoside chemical induction, but blue light induction[1].
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We chose blue light induction primarily because chemically induced gene expression systems are valuable tools for controlling biological processes for applications in basic science and biotechnology. While allowing tunability and some degree of spatial control, these systems have some limitations -they are unable to achieve complex spatiotemporal regulation [2], and often lack reversibility or require washing steps to achieve it [3]. These limitations can be overcome by using light, rather than small molecules, as external triggers. Under the light, for example, pulsating inputs that alternate between dark (off) and maximum intensity (fully on) can be produced [4] and have been shown to lead to effects not achievable with graded intensity light, such as reduced cell-to-cell variability in gene expression [5]. Indeed, the amount of cell-to-cell variation can be adjusted by adjusting the duty cycle, defined as the fraction of time that light is fully on, providing a new mode of control for studying stochasticity in gene expression[6]. This type of pulsatile input has also recently been shown to enhance the biosynthesis of products in engineered cells, enabling a new type of bioreactor operation[7]. The enzyme expression was adjusted to increase the fermentation yield [8]. This is a great help for the design of our photo-controlled herbicide production.
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===Build===
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According to our design, the AraC and ParaBAD genes of the Arabinose induction and regulation system from Escherichia coli and the vivid gene from Streptomyces were synthesized respectively. eSD was added as the ribosome binding site. The synthetic genes were amplified by PCR, and the gene fragments were connected by golden gate according to the circuit diagram design.
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We selected Native Pc, J23101, and porin as operon gene promoters, and determined the best promoters by synthesizing and detecting the final thallus concentration and the expression yield of the green fluorescent protein.
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[[File:XJTU-3.png|200px|]]
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FIG.3 PCR electrophoretic diagram of VVDAraC chimera gene
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[[File:XJTU-6.png|200px]]
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FIG.4 PCR electrophoretic diagram of PAVVDH-J23101 colony
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[[File:XJTU-7.png|200px]]
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FIG.5 PCR electrophoretic diagram of PAVVDH-porin colony
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==References==
 
[1] ROMANO E, BAUMSCHLAGER A, AKMERIÇ E B, et al. Engineering AraC to make it responsive to light instead of arabinose [J]. Nat Chem Biol, 2021, 17(7): 817-27.
 
[1] ROMANO E, BAUMSCHLAGER A, AKMERIÇ E B, et al. Engineering AraC to make it responsive to light instead of arabinose [J]. Nat Chem Biol, 2021, 17(7): 817-27.
 +
 
[2] RAMAKRISHNAN P, TABOR J J. Repurposing Synechocystis PCC6803 UirS-UirR as a UV-Violet/Green Photoreversible Transcriptional Regulatory Tool in E. coli [J]. ACS Synth Biol, 2016, 5(7): 733-40.
 
[2] RAMAKRISHNAN P, TABOR J J. Repurposing Synechocystis PCC6803 UirS-UirR as a UV-Violet/Green Photoreversible Transcriptional Regulatory Tool in E. coli [J]. ACS Synth Biol, 2016, 5(7): 733-40.

Latest revision as of 13:49, 10 October 2022


VVDH


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 223
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 223
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 223
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 223
  • 1000
    COMPATIBLE WITH RFC[1000]


Profile

Name:VVD

Base Pairs:453bp

Origin:Neurospora crassa

Performed E. coli codon optimization

Usage&Biology

FIG. 1 Schematic diagram of blue light induction regulatory system
FIG. 2 Schematic diagram of blue light-induced regulation system

Research

To realize the release of light-controlled products to achieve the goal of intelligent and sustained release, after an extensive literature survey, we found that the arabinose operon was modified to manipulate downstream gene expression, not through arabinoside chemical induction, but blue light induction[1].

We chose blue light induction primarily because chemically induced gene expression systems are valuable tools for controlling biological processes for applications in basic science and biotechnology. While allowing tunability and some degree of spatial control, these systems have some limitations -they are unable to achieve complex spatiotemporal regulation [2], and often lack reversibility or require washing steps to achieve it [3]. These limitations can be overcome by using light, rather than small molecules, as external triggers. Under the light, for example, pulsating inputs that alternate between dark (off) and maximum intensity (fully on) can be produced [4] and have been shown to lead to effects not achievable with graded intensity light, such as reduced cell-to-cell variability in gene expression [5]. Indeed, the amount of cell-to-cell variation can be adjusted by adjusting the duty cycle, defined as the fraction of time that light is fully on, providing a new mode of control for studying stochasticity in gene expression[6]. This type of pulsatile input has also recently been shown to enhance the biosynthesis of products in engineered cells, enabling a new type of bioreactor operation[7]. The enzyme expression was adjusted to increase the fermentation yield [8]. This is a great help for the design of our photo-controlled herbicide production.





Build

According to our design, the AraC and ParaBAD genes of the Arabinose induction and regulation system from Escherichia coli and the vivid gene from Streptomyces were synthesized respectively. eSD was added as the ribosome binding site. The synthetic genes were amplified by PCR, and the gene fragments were connected by golden gate according to the circuit diagram design. We selected Native Pc, J23101, and porin as operon gene promoters, and determined the best promoters by synthesizing and detecting the final thallus concentration and the expression yield of the green fluorescent protein.

XJTU-3.png

FIG.3 PCR electrophoretic diagram of VVDAraC chimera gene

XJTU-6.png

FIG.4 PCR electrophoretic diagram of PAVVDH-J23101 colony

XJTU-7.png

FIG.5 PCR electrophoretic diagram of PAVVDH-porin colony

References

[1] ROMANO E, BAUMSCHLAGER A, AKMERIÇ E B, et al. Engineering AraC to make it responsive to light instead of arabinose [J]. Nat Chem Biol, 2021, 17(7): 817-27.

[2] RAMAKRISHNAN P, TABOR J J. Repurposing Synechocystis PCC6803 UirS-UirR as a UV-Violet/Green Photoreversible Transcriptional Regulatory Tool in E. coli [J]. ACS Synth Biol, 2016, 5(7): 733-40.