Difference between revisions of "Part:BBa K2739002:Design"

 
 
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===Design Notes===
 
===Design Notes===
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This part was designed to investigate its relation with phasin.  
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This composite part is consisted PhaR. This part was designed to allow the investigation of its relation and PHA production through PHA operon with and without phasin. Sequence of PhaR and its native promoter could be found in iGEM registry, as a basic parts (BBa_K108015) and being not available. The source of this sequences were recorded as 1. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology. 2002, 148:2413–2426. and 2. NCBI (with no further information), having no mentioned if the sequence was codon optimised.
  
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We access the online NCBI data and found out the current available phaR sequence (AM260479.1) is being 4 bp different to BBa_K108015. Therefore we have used the new phaR and promoter sequence as our new template. Due to the excessive GC in the sequence, we have it codon optimised to E.coli and generated the part through the IDT company service.
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https://static.igem.org/mediawiki/parts/c/cd/T--Edinburgh_OG_BBa_K2739000--image_9.jpeg
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Figure 1. Comparison of phaR sequence from  iGEM08_Tsinghua (BBa_K108015) and online NCBI(AM260479.1). Capital letters: NCBI(AM260479.1)  and Small letter: iGEM08_Tsinghua (BBa_K108015).
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https://static.igem.org/mediawiki/parts/9/9a/T--Edinburgh_OG_BBa_K2739000--image_10.jpeg
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Figure 2. Comparison of phaR sequence before and after codon optimisation. Capital letters:  NCBI (AM260479.1) and Small letter: codon optimised version (BBa_K2739001).
  
  
 
===Source===
 
===Source===
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1. BBa_K108015 and NCBI (AM260479.1)
  
−
This part is not present in the iGEM before and have to generated through the idt company service. The sequence is obtained from previous publication, which investigated R.eutropha and its gene for PHA production.
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2. ProR and PhaR Sequence selection based on Reference 1 information.  
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3. PhaR sequence codon optimised through IDT
  
 
===References===
 
===References===
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1. Yamada M, Takahashi S, Okahata Y, Doi Y, Numata K. (2013 ) Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]. AMB Express. 3(1):6.

Latest revision as of 01:18, 18 October 2018


Codon optimizated PhaR (The Phasin autoregulation system)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 147


Design Notes

This composite part is consisted PhaR. This part was designed to allow the investigation of its relation and PHA production through PHA operon with and without phasin. Sequence of PhaR and its native promoter could be found in iGEM registry, as a basic parts (BBa_K108015) and being not available. The source of this sequences were recorded as 1. Regulation of phasin expression and polyhydroxyalkanoate (PHA) granule formation in Ralstonia eutropha H16. Microbiology. 2002, 148:2413–2426. and 2. NCBI (with no further information), having no mentioned if the sequence was codon optimised.

We access the online NCBI data and found out the current available phaR sequence (AM260479.1) is being 4 bp different to BBa_K108015. Therefore we have used the new phaR and promoter sequence as our new template. Due to the excessive GC in the sequence, we have it codon optimised to E.coli and generated the part through the IDT company service.


T--Edinburgh_OG_BBa_K2739000--image_9.jpeg

Figure 1. Comparison of phaR sequence from iGEM08_Tsinghua (BBa_K108015) and online NCBI(AM260479.1). Capital letters: NCBI(AM260479.1) and Small letter: iGEM08_Tsinghua (BBa_K108015).

T--Edinburgh_OG_BBa_K2739000--image_10.jpeg

Figure 2. Comparison of phaR sequence before and after codon optimisation. Capital letters: NCBI (AM260479.1) and Small letter: codon optimised version (BBa_K2739001).


Source

1. BBa_K108015 and NCBI (AM260479.1)

2. ProR and PhaR Sequence selection based on Reference 1 information.

3. PhaR sequence codon optimised through IDT

References

1. Yamada M, Takahashi S, Okahata Y, Doi Y, Numata K. (2013 ) Monitoring and kinetic analysis of the molecular interactions by which a repressor protein, PhaR, binds to target DNAs and poly[(R)-3-hydroxybutyrate]. AMB Express. 3(1):6.