Difference between revisions of "Part:BBa K4182000"

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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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[[File:XJTU-1.png|300px|thumb|left|FIG. 1 Principle of blue light induction regulatory system]]
  
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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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[[File:XJTU-2.png|300px|thumb|left|FIG. 2 Principle of blue light-induction regulation system]]
  
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===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.
  
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===Build===
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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.
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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 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.  
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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.We selected sfGFP as the reporter to verify the regulation of the system. In order to test the effect of VVD-AraC expression level on the downstream gene expression, three promoters-native Pc, J23101 and porin promoter was selected in our study (BBa_K4182001, BBa_K4182002, BBa_K4182003). The blue-light inducible circuit is shown as follows (Figure 3).
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[[File:XJTU-Design1.png|500px]]
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FIG. 3 The blue light-induced circuit
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According to our design, the VVD gene from Streptomyces were chemically synthesized, and the AraC-ParaBAD promoter in arabinose operon was amplified from Escherichia coli, and eSD from E. coli was served as the ribosome binding site. The three promoters-native Pc, J23101, and porin was obtained by PCR. All the fragments were ligated into pBBRMCS1 vector in one step via Golden Gate Assembly. Figures 4 and 5 shows the PCR fragments used for the circuit construction. The recombinant plasmids were verified by colony PCR as shown in Figure 6, which are further confirmed by sequencing. As a result, three plasmids PVVDH-Pc, PVVDH-J23101, PVVDH-porin, were successfully constructed for further test including cell growth and the expression of GFP.  
  
 
[[File:XJTU-3.png|200px|]]
 
[[File:XJTU-3.png|200px|]]
  
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FIG.3 PCR electrophoretic diagram of VVDAraC chimera gene
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FIG.4 PCR result of J23101-VVD fragment
  
 
[[File:XJTU-6.png|200px]]
 
[[File:XJTU-6.png|200px]]
  
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FIG.4 PCR electrophoretic diagram of PAVVDH-J23101 colony
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FIG.5 PCR result of porin-VVD fragment
  
 
[[File:XJTU-7.png|200px]]
 
[[File:XJTU-7.png|200px]]
  
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FIG.5 PCR electrophoretic diagram of PAVVDH-porin colony
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FIG.6 Colony PCR verification of plasmid PAVVDH-porin
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[[File:XJTU-bl1.png|500px]]
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FIG.7 mRNA level of VVD under different promoters without blue light
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[[File:XJTU-VVD8.png|300px]]
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FIG.8 Microscopy observation of strain harboring VVD after blue-light induction
  
 
==References==
 
==References==

Latest revision as of 19:35, 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 Principle of blue light induction regulatory system
FIG. 2 Principle of blue light-induction regulation system


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.

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.We selected sfGFP as the reporter to verify the regulation of the system. In order to test the effect of VVD-AraC expression level on the downstream gene expression, three promoters-native Pc, J23101 and porin promoter was selected in our study (BBa_K4182001, BBa_K4182002, BBa_K4182003). The blue-light inducible circuit is shown as follows (Figure 3).

XJTU-Design1.png

FIG. 3 The blue light-induced circuit


According to our design, the VVD gene from Streptomyces were chemically synthesized, and the AraC-ParaBAD promoter in arabinose operon was amplified from Escherichia coli, and eSD from E. coli was served as the ribosome binding site. The three promoters-native Pc, J23101, and porin was obtained by PCR. All the fragments were ligated into pBBRMCS1 vector in one step via Golden Gate Assembly. Figures 4 and 5 shows the PCR fragments used for the circuit construction. The recombinant plasmids were verified by colony PCR as shown in Figure 6, which are further confirmed by sequencing. As a result, three plasmids PVVDH-Pc, PVVDH-J23101, PVVDH-porin, were successfully constructed for further test including cell growth and the expression of GFP.

XJTU-3.png

FIG.4 PCR result of J23101-VVD fragment

XJTU-6.png

FIG.5 PCR result of porin-VVD fragment

XJTU-7.png

FIG.6 Colony PCR verification of plasmid PAVVDH-porin

XJTU-bl1.png

FIG.7 mRNA level of VVD under different promoters without blue light

XJTU-VVD8.png

FIG.8 Microscopy observation of strain harboring VVD after blue-light induction

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.