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

Latest revision as of 12:53, 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]

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

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

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 ===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===