Difference between revisions of "Part:BBa K4271001:Design"

 
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DNA sequence of the OPH is optimized for the protein expression on E. coli.
 
DNA sequence of the OPH is optimized for the protein expression on E. coli.
  
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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. In the process of paraoxon degradation, our target gene oph encodes for the enzyme organophosphate hydrolase (OPH), which hydrolyzes paraoxon into dimethyl phosphate (DMP) and p-nitrophenol (pNP) (Fig.1). Since the E.coli bacterial strain BL21 (DE3) has a high level protein expression with T7 RNA polymerase, we chose it as a host cell for our experiment. The vector we used is pET-22b, which includes a T7 promoter (BBa_I712074), lac operator (BBa_K2406019), RBS (BBa_K2924053), OPH gene (BBa_K4271000), and T7 terminator (BBa_K731721) (Fig.6). We also included a pelB signal peptide, which plays a significant role in our experiment by directing our target OPH enzyme to the bacterial periplasm, thereby enhancing the enzyme’s activity at the specific location (Jain, Monika et al.). 
  
  

Revision as of 12:48, 5 October 2022


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


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]


Design Notes

DNA sequence of the OPH is optimized for the protein expression on E. coli.

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. In the process of paraoxon degradation, our target gene oph encodes for the enzyme organophosphate hydrolase (OPH), which hydrolyzes paraoxon into dimethyl phosphate (DMP) and p-nitrophenol (pNP) (Fig.1). Since the E.coli bacterial strain BL21 (DE3) has a high level protein expression with T7 RNA polymerase, we chose it as a host cell for our experiment. The vector we used is pET-22b, which includes a T7 promoter (BBa_I712074), lac operator (BBa_K2406019), RBS (BBa_K2924053), OPH gene (BBa_K4271000), and T7 terminator (BBa_K731721) (Fig.6). We also included a pelB signal peptide, which plays a significant role in our experiment by directing our target OPH enzyme to the bacterial periplasm, thereby enhancing the enzyme’s activity at the specific location (Jain, Monika et al.).


Source

pET22b vector, T7 bacteriophage, OPH from phosphotriesterase [Agrobacterium tumefaciens]

References