Part:BBa_K4271001:Design
T7 Promoter + Lac operator + RBS + OPH + T7 terminator
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1259
Illegal NotI site found at 1219 - 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1228
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE 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 - 1000COMPATIBLE 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]