Part:BBa_K2244011
CloE promoter+TorA+opdA+T1 terminator
The device is a functional composite part containing a coding sequence opdA (BBa_K215090) with a TorA signal peptide fused to its N-terminus for protein export (BBa_K2244003).
Biology -Co1E promoter (BBa_) is derived from the promoter region of colicin E gene located in the ColE1 plasmid of E.coli. ColE promoter contains a ‘SOS’ operator region that allows the binding of LexA protein to repress transcription. DNA-binding component of LexA repressor in LEV1 would form a dimer and bind to the operator sequence thus halts the activity of ColE promoter. - TorA-opdA contains a signal peptide fused to the N-terminus of opdA. opdA encodes organophosphate hydrolase (OPH) which is a homodimeric organophosphate triesterase that requires metal ion as a cofactor to degrade a wide range of toxic organophosphates. OPH can hydrolyze various phosphorus-ester bonds including P-O, P-F, P-CN, and P-S bonds. TorA is an E. coli twin-arginine signal peptide bearing a consensus motif of SRRxFLK. TorA-opdA allows exportation of opdA encoding protein to periplasmic space. -T1 terminator (BBa_), it is the most used terminator in E. coli system.
Usage In our project this year, this device worked in the lightOFF system (BBa_k2244009) to replace the ColE promoter+mCherry+T1 terminator section and to allow the expression of periplasmic OPH when induced in darkness. To demonstrated the functionality of this part, we performed ELISA studies, enzymatic activity and whole cell stability studies (Figure 1-3) to prove that functional OPH was successfully secreted in periplasmic.
Figure: ELISA studies of OPH protein expression in periplasmic fraction, cytoplasmic fraction, whole cell, and periplasmic fraction of pLEV1(408) (control strain).
Figure: Specific OPH activities of whole cell, periplasmic fraction, cytoplasmic fraction and control periplasmic fraction (lightOFF). The activity was assayed with paraoxon as substrate. Data are mean values+/-standard derivations from three replicates.
Figure illustration of OPH activity vs various concentrations of periplasmic fractions from OPH-expressed cell strain (black) and control strain (Red). The activity was assayed with paraoxon as substrate. Data are mean values+/-standard derivations from three replicates.
Reference
1) Bulina, M. E., Chudakov, D. M., Britanova, O. V. & Lukyanov. K.. 2003. A genetically encoded photosensitizer. Nat. Biotechnol. 24, 95-99. 2)Tour, O., Meijer, R. M., Zacharias, D. A., Adams, S. R. & Tsien, R. Y, 2003. Genetically targeted chromophore-assisted light inactivation. Nat. Biotechnol. 21, 1505–1508. 3)Wong, E. V., David, S., Jacob, M. H. & Jay, D. G, 2003. Inactivation of myelin-associated glycoprotein enhances optic nerve regeneration. J. Neurosci. 23, 3112–3117. 4)Takemoto, K., Matsuda, T., Sakai, N., Fu, D., Noda, M., Uchiyama, S., Kotera, I., Arai, Y., Horiuchi, M., Fukui, K. and Ayabe, T., 2013. SuperNova, a monomeric photosensitizing fluorescent protein for chromophore-assisted light inactivation. Scientific reports.
The device we constructed this year is one of the functional plasmids in the light-regulate gene expression system. With torA-opdA(BBa_K2244003) which is constructed in the other device. it’s can be successful expressed by using light regulate system.that can encoding organophosphorus hydrolase(OPH) to degrade the pesitcide residuce.
To enable secretion of OPH (gene product of opdA) to the periplasm of E. coli for the development of live cell biocatalysts, the TorA signal peptide followed by four amino acid residues of the mature TorA protein is fused directly to the N-terminal of OPH domain. TorA signal peptide contains a twin-arginine motif of ‘SRRxFLA’, and a recognition site for type I signal peptidases.
Biology
ColE promoter(BBa_K2244006) sequence is derived from the promoter region of colicin E gene located in the ColE1 plasmid of E.coli. ColE promoter contains a ‘SOS’ operator region that allows the binding of LexA protein to repress transcription.opdA gene was obtained from the chromosome of Agrobacterium radiobacter. The enzyme opdA encodes is a transposable phosphotriesterase. In addition, opdA-like gene is evolutionary conserved in many bacteria species, i.e. Sphingobium fuliginis, Brevundimonas diminuta, and Mycobacterium genera, which indicates horizontal mobility.
TorA(BBa_K2244003) is a twin-arginine signal peptide from E. coli. The twin-arginine system a bacterial protein export pathway with the remarkable ability to transport folded proteins across the cytoplasmic membrane. Proteins, with the N-terminal TorA bearing a consensus motif of SRRxFLK, are targeted to a membrane-embedded Tat translocase. T1terminator is the most commonly used terminator. It seems to be reliable.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 1286
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 383
Illegal AgeI site found at 578
Illegal AgeI site found at 917 - 1000COMPATIBLE WITH RFC[1000]
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