Part:BBa_K4271001

T7 Promoter + Lac operator + RBS + OPH + T7 terminator

This sequence is responsible for the regulation and expression of the OPH gene. The T7 promoter, transcribed by only the T7 RNA polymerase, identifies the sequence downstream and enables fast and effective transcription. We further designed a lac operon, so that upon IPTG induction, the lacI repressor protein will be detached from the lacI gene, leading to the transcription and translation of our target oph gene. The ribosome binding site (RBS) is where the ribosome bind on the mRNA for translation. This RBS is taken from the pET22B vector. OPH a gene that encodes organophosphate hydrolase, paraoxon, a type of organic phosphate and insecticide. The product of the paraoxon degradation, pNP, will be detected to evaluate the efficacy of the gene. The T7 terminator identifies the end of the transcription sequence.

Usage and Biology

Purity issues (update):

Synthetic oph gene we used in this study is derived from the opd (organophosphate degradation) gene in Agrobacterium tumefaciens and performed with codon usage optimization for E. coli heteroexpression. We digested the oph gene with BamHI and HindIII, subcloned it to pET22b vectors that underwent the same restriction enzyme digestion, then transformed the recombinant into E. coli DH5α. The transformation was conducted by plasmid extraction through mini-prep.

We later confirmed the insertion of our oph gene into the enzyme plasmid by enzyme digestion, cutting the recombinant DNA with BamHI and HindIII respectively, and observing the same band sizes of 6.5 kilobases after gel electrophoresis (Fig.7). We later digested our pET22b::OPH again with both BamHI and HindIII, two of resulting DNA bands include the 1071 base-long oph and the 5479 base-long pET22b vector (Fig.8). Finally, the plasmid was transformed into the competent cells E.coli BL21(DE3) via heat shock, which we later used to examine the level of paraoxon degradation by our enzyme plasmid.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 1259
  Illegal NotI site found at 1219
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 1228
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 330
  Illegal AgeI site found at 150
  Illegal AgeI site found at 435
  Illegal AgeI site found at 570
  Illegal AgeI site found at 633
  
• 1000
  COMPATIBLE WITH RFC[1000]