Difference between revisions of "Part:BBa K4182002:Design"

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==Design Notes==
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==Profile==
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===Base Pairs===
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894
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===Design Notes===
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The codon of E. coli was optimized
 
The codon of E. coli was optimized
  
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E.coli&Neurosparo ceassa
 
E.coli&Neurosparo ceassa
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==Usage&Test==
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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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Although the J23101 promoter is not the best, we still want to share our work results.
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[[File:XJTU-3.png|300px]]
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FIG.1 PCR electrophoretic diagram of VVDAraC chimera gene
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===Test===
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It can be seen from Figures above that porin has a higher VVD transcription level and sfGFP background expression than the J23101 promoter under non-blue light induction, indicating that the porin promoter can better and more precisely initiate and regulate gene expression. FIG. 17 further proves that porin has a larger dynamic response range and better sensitivity when induced by blue light than the native PC promoter and J23101 promoter. Therefore, the PAVVDH-porin promoter was selected as the follow-up research object.
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[[File:XJTU-bl1.png|500px]]
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FIG.4 mRNA level of VVD and sfGFP under different promoters without blue light
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[[File:XJTU-bl2.png|500px]]
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FIG.5 Differential expression of green fluorescent protein of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin
  
 
===References===
 
===References===

Revision as of 14:49, 10 October 2022


J23101-eSD-VVD-AraC


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 634
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 28
    Illegal PstI site found at 634
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 634
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 634
  • 1000
    COMPATIBLE WITH RFC[1000]


Profile

Base Pairs

894

Design Notes

The codon of E. coli was optimized

Source

E.coli&Neurosparo ceassa

Usage&Test

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.

Although the J23101 promoter is not the best, we still want to share our work results.

XJTU-3.png

FIG.1 PCR electrophoretic diagram of VVDAraC chimera gene


Test

It can be seen from Figures above that porin has a higher VVD transcription level and sfGFP background expression than the J23101 promoter under non-blue light induction, indicating that the porin promoter can better and more precisely initiate and regulate gene expression. FIG. 17 further proves that porin has a larger dynamic response range and better sensitivity when induced by blue light than the native PC promoter and J23101 promoter. Therefore, the PAVVDH-porin promoter was selected as the follow-up research object.

XJTU-bl1.png

FIG.4 mRNA level of VVD and sfGFP under different promoters without blue light

XJTU-bl2.png

FIG.5 Differential expression of green fluorescent protein of PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin

References