Difference between revisions of "Part:BBa K4182000"

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===Research===
 
===Research===
  
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Compared to chemical induction, the light induction is more fine-tuned and spatial controllable. In order to achieve blue-light induction, we replace the arabinose binding and dimerization domain with blue-light responsive VVD domain, generating VVD-AraC fusion protein, which will dimerization under light and promote the downstream PBAD promoter.
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To realize the controllable synthesis and release of products, after an extensive literature survey, we found that the arabinose operon can be engineered to regulated downstream gene expression by blue light induction instead of arabinose chemical induction.
 +
 
 +
Chemically induced gene expression systems are valuable tools to control biological processes for applications in basic science and biotechnology. While for the tuned and spatial control of gene expression, chemically induced systems have some limitations-they are unable to achieve complex spatiotemporal regulation, and often lack reversibility or require washing steps to achieve it. These limitations can be overcome by using light, rather than small molecules, as external triggers. This type of pulsatile input has also recently been found to enhance the biosynthesis of products in engineered cells, enabling a new type of bioreactor operation that is much easier to handle than chemical induction. The enzyme expression was adjusted to increase the fermentation yield.
 +
 
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Based on the above information, we replace the arabinose binding and dimerization domain with blue-light responsive VVD domain, generating VVD-AraC fusion protein, which will dimerization under light and promote the downstream PBAD promoter.
  
 
===Build===
 
===Build===

Revision as of 18:58, 13 October 2022


VVDH

The gene encodes blue-light responsive protein VVD. It is a truncated VVD without N terminus 35aa.

Sequence and Features


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 controllable synthesis and release of products, after an extensive literature survey, we found that the arabinose operon can be engineered to regulated downstream gene expression by blue light induction instead of arabinose chemical induction.

Chemically induced gene expression systems are valuable tools to control biological processes for applications in basic science and biotechnology. While for the tuned and spatial control of gene expression, chemically induced systems have some limitations-they are unable to achieve complex spatiotemporal regulation, and often lack reversibility or require washing steps to achieve it. These limitations can be overcome by using light, rather than small molecules, as external triggers. This type of pulsatile input has also recently been found to enhance the biosynthesis of products in engineered cells, enabling a new type of bioreactor operation that is much easier to handle than chemical induction. The enzyme expression was adjusted to increase the fermentation yield.

Based on the above information, we replace the arabinose binding and dimerization domain with blue-light responsive VVD domain, generating VVD-AraC fusion protein, which will dimerization under light and promote the downstream PBAD promoter.

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 promoters for VVD-araC protein expression, and determined the best promoters by the expression yield of the green fluorescent protein. Also see more results of blue-light and VVD regulated sfGFP expression in parts BBa_K4182001 and BBa_K4182002.

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.