Part:BBa_K2010005:Design
ompT + sfGFP + PETase (PET-degrading enzyme, origin I. sakaiensis)
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 809
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 1227
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.
The ompT tag was included for excretion of PETase.
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).