Difference between revisions of "Part:BBa K2244011"
 
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The device we constructed this year is one of the functional plasmids in the light-regulate gene expression system. With torA-opdA([https://parts.igem.org/Part:BBa_K2244008 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.  
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The device is a functional composite part containing a coding sequence opdA ([https://parts.igem.org/Part:BBa_K215090 BBa_K215090]) with a TorA signal peptide fused to its N-terminus for protein export ([https://parts.igem.org/Part:BBa_K2244003 BBa_K2244003]).
  
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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===
 
===Biology===
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ColE promoter([https://parts.igem.org/Part:BBa_K2244006 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.
 
  
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TorA([https://parts.igem.org/Part:BBa_K2244003 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.  
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-ColE promoter ([https://parts.igem.org/Part:BBa_K2244006 BBa_K2244006]) 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.
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T1terminator is the most commonly used terminator. It seems to be reliable.
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- TorA-opdA([https://parts.igem.org/Part:BBa_K2244003 BBa_K2244003]) 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.
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-T1 terminator ([https://parts.igem.org/Part:BBa_B0010 BBa_B0010]), it is the most used terminator in E. coli system.
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===Usage===
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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.
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<center><b>Figure 1:</b> ELISA studies of OPH protein expression in periplasmic fraction, cytoplasmic fraction, whole cell, and periplasmic fraction of pLEV1(408) (control strain).</center>
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<center><b>Figure 2:</b> 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.</center>
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<center><img src="https://static.igem.org/mediawiki/parts/c/c3/2121541.png" style=" width:85%" /> </center>
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<center><b>Figure 3:</b> 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.</center>
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===Reference===
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1) Bulina, M. E., Chudakov, D. M., Britanova, O. V. & Lukyanov. K.. 2003. A genetically encoded photosensitizer. Nat. Biotechnol. 24, 95-99.
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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.
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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.
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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.
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Latest revision as of 09:35, 28 October 2017

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

-ColE promoter (BBa_K2244006) 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(BBa_K2244003) 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_B0010), 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 1: ELISA studies of OPH protein expression in periplasmic fraction, cytoplasmic fraction, whole cell, and periplasmic fraction of pLEV1(408) (control strain).


Figure 2: 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 3: 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.



Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal XhoI site found at 1286
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 383
    Illegal AgeI site found at 578
    Illegal AgeI site found at 917
  • 1000
    COMPATIBLE WITH RFC[1000]