Difference between revisions of "Part:BBa K2244011"
 
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The device is a functional plasmid containing a light repressor LEV1 ([https://parts.igem.org/Part:BBa_K2244005 BBa_K2244005]), which undergoes conformational change upon light irradiation and dimerized to bind regulatory sequence on ColE promoter ([https://parts.igem.org/Part:BBa_K2244006 BBa_K2244006]), thus inhibits target gene expression. While in darkness, dimerization does not occur thus gene expression proceeds. This device is a light repressing system with high induction efficiency and low leakage. mCherry (([https://parts.igem.org/Part:BBa_K2244008 BBa_K2244008])) is reporter gene used in this system to prove its function as well as help to characterize it. mCherry gene can be replaced by any gene sequence in light-regulated studies
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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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===Biology===
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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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- 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><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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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K2244011 SequenceAndFeatures</partinfo>
  
  
 
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===Functional Parameters===
 
===Functional Parameters===
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<partinfo>BBa_K2244011 parameters</partinfo>
 
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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]