Difference between revisions of "Part:BBa K2010004:Design"

 
 
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===Design Notes===
 
===Design Notes===
−
The DNA sequence has been optimized for E. coli K12 and modified to facilitate gBlock synthesis, by avoiding repeated sequences.
+
This sequence was optimized for E. coli K12 using the Codon Optimization Tool from the IDT website.
  
 +
The T7 promoter was used in order to have strong, inducible transcription, in conjunction with NEB’s T7 Express lysY/Iq Competent E. coli.
  
 +
RBS 34 was used for standard efficiency.
 +
 +
The GGS linker was used to prevent the His-tag from interfering with the folding of PETase.
 +
 +
The His-tag was included for His-tag purification.
 +
 +
sfGFP was included as a reporter protein.
  
  
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===Source===
 
===Source===
  
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This sequence is publicly known and was privately modified.  
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T7 promoter: BBa_I712074
 +
 
 +
RBS: BBa_B0034
 +
 
 +
PETase, originally named ISF6_4831, comes from Ideonella sakaiensis, identified in "A bacterium that degrades and assimilates poly(ethylene terephthalate)" (Yoshida et al. 2016).
  
 
===References===
 
===References===

Latest revision as of 07:15, 8 October 2016


sfGFP + PETase (PET-degrading enzyme, origin I. sakaiensis)


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 743
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 1161


Design Notes

This sequence was optimized for E. coli K12 using the Codon Optimization Tool from the IDT website.

The T7 promoter was used in order to have strong, inducible transcription, in conjunction with NEB’s T7 Express lysY/Iq Competent E. coli.

RBS 34 was used for standard efficiency.

The GGS linker was used to prevent the His-tag from interfering with the folding of PETase.

The His-tag was included for His-tag purification.

sfGFP was included as a reporter protein.


Source

T7 promoter: BBa_I712074

RBS: BBa_B0034

PETase, originally named ISF6_4831, comes from Ideonella sakaiensis, identified in "A bacterium that degrades and assimilates poly(ethylene terephthalate)" (Yoshida et al. 2016).

References