Difference between revisions of "Part:BBa K2244010"
 
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The device is a functional plasmid containing a suicide gene supernova(BBa_K1491017), which is a mutant form of KillerRed(BBa_K1184000), the first genetically-encoded photosensitizer. Upon illumination, reactive oxygen species (ROS) are generated to induce cell apoptosis , We incorporated into our lightOFF system (BBa_K2244009) so that supernova can be induced fully in darkness and start secreting ROS upon light irradiation to promote cell death.
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The device is a functional composite part containing a suicide gene supernova ([https://parts.igem.org/Part:BBa_K1491017 BBa_K1491017]).
  
  
 
===Biology===
 
===Biology===
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LEV1 repressor is a fusion protein of VVD and LexA, LexA repressor is a transcriptional repressor of SOS regulon in E.coli. It’s form chromosome of Escherichia coli str. K-12 substr. MG1655 (strain: K-12, substrain: MG1655) LexA autocleavage, stimulated by RecA, of the first 84 aa of LexA removes the DNA binding region and is required to activate the SOS response.  LexA is a protein that belongs to the LexA family .
 
  
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Blue light sensor VIVID was derived from the chromosome of Neurospora crassa. The LOV domain of the protein VVD has the capacity to self-dimerize upon light stimulation, Based on this property, the VVD LOV domain was fused with a smaller version of the Gal4 DNA binding domain and the p65 transactivation domain. A common feature of several blue light photoreceptors is the presence of LOV domains, which are able to bind a molecule of flavin mononucleotide (FMN) or flavin adenine dinucleotide (FAD) as chromophore, forming upon light stimulation a cysteinyl flavin C4a adduct.
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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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Supernova is a mutant of killer red (BBa_K1184000),KillerRed is a red fluorescent protein that produces reactive oxygen species (ROS) in the presence of yellow-orange light (540-585 nm). KillerRed is engineered from anm2CP to be phototoxic. KillerRed is spectrally similar to mRFP1 with a similar brightness. KillerRed is oligomeric and may form large aggregates in cells.  
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-Supernova([https://parts.igem.org/Part:BBa_K1491017 BBa_K1491017]) encodes a phototoxic compound, which is a mutant form of KillerRed([https://parts.igem.org/Part:BBa_K1184000 BBa_K1184000]), the first genetically-encoded photosensitizer. Upon illumination, reactive oxygen species (ROS) are generated to induce cell apoptosis. KillerRed is engineered from anm2CP to be phototoxic.  
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-LEV1 repressor ([https://parts.igem.org/Part:BBa_K2244005 BBa_K2244005]) is a fusion protein of VVD and LexA. Blue light sensor VIVID was derived from the chromosome of Neurospora crassa. The LOV domain of the protein VVD has the capacity to self-dimerize upon light stimulation. LexA repressor is a transcriptional repressor of SOS regulon in E.coli. LEV1 is the core component of this device.
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-Constitutive promoter ([https://parts.igem.org/Part:BBa_K2244012 BBa_K2244012]), in this device, it is used to constitutively express Lev1 gene.
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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) by replacing mCherry gene with supernova. This is to allow supernova to be firstly induced in darkness and then secrete ROS upon light illumination which promotes cell death (Figure 1-2). This allows a good incorperation of suicide system into our light-regulated expression system.
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<html>
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<body>
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<center><img src="https://static.igem.org/mediawiki/parts/4/42/Superino.png" style=" width:70%" /> </center>
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</body>
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</html>
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<center><b>Figure 1:</b> The apoptosis effect of supernova to E. coli cells. Light exposure greatly reduced the CFU number of bacteria cells, while darkness exposure allowed normal growth. Inert: supernova expression indicated by pink cells.
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</center>
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<hr/>
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<html>
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<body>
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<center><img src="https://static.igem.org/mediawiki/parts/archive/f/f3/20171029021808%21Daark1111.png" style=" width:70%" /> </center>
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</body>
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</html>
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<center><b>Figure 2:</b> The apoptosis effect of supernova to E. coli cells. Light exposure greatly reduced the CFU number of bacteria cells, while darkness exposure allowed normal growth during a 48-h period of time. LB plates were grown for 24 h after light irradation/kept in darkenss.  </center>
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===Improvement===
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This part is an improved part of supernova ([https://parts.igem.org/Part:BBa_K1491017 BBa_K1491017]). The improvement was made by inserting supernova gene into a light-regulated expression system, lightoff ([https://parts.igem.org/Part:BBa_K2244009 BBa_K2244009]), cell apoptosis can thus be induced by firstly expressing supernova gene in darkness, and then expose cells to light illumination that results to ROS release and the subsequent cell death. The entire process is purely regulated by light. We believe this has improved the original use of supernova in a T7 system using IPTG induction, which requires additional chemical inducer.This improvement has made the use of supernova suicide system more convenient and more easily manipulated.
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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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This sequence is codon optimized for mammalian cells and has 13 rare proline codons for E. coli (CCC) and one rare arginine codon (AGA). Codon optimization should be taken into consideration if large amounts of the protein are required. Expression on a high-copy plasmid has produced detectable fluorescent signal, however.
 
  
  
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===Usage and Biology===
 
  
 
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Latest revision as of 02:18, 31 October 2017

ColE promoter +supernova gene +Lev1 gene


The device is a functional composite part containing a suicide gene supernova (BBa_K1491017).


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.

-Supernova(BBa_K1491017) encodes a phototoxic compound, which is a mutant form of KillerRed(BBa_K1184000), the first genetically-encoded photosensitizer. Upon illumination, reactive oxygen species (ROS) are generated to induce cell apoptosis. KillerRed is engineered from anm2CP to be phototoxic.

-LEV1 repressor (BBa_K2244005) is a fusion protein of VVD and LexA. Blue light sensor VIVID was derived from the chromosome of Neurospora crassa. The LOV domain of the protein VVD has the capacity to self-dimerize upon light stimulation. LexA repressor is a transcriptional repressor of SOS regulon in E.coli. LEV1 is the core component of this device.

-Constitutive promoter (BBa_K2244012), in this device, it is used to constitutively express Lev1 gene.

-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) by replacing mCherry gene with supernova. This is to allow supernova to be firstly induced in darkness and then secrete ROS upon light illumination which promotes cell death (Figure 1-2). This allows a good incorperation of suicide system into our light-regulated expression system.

Figure 1: The apoptosis effect of supernova to E. coli cells. Light exposure greatly reduced the CFU number of bacteria cells, while darkness exposure allowed normal growth. Inert: supernova expression indicated by pink cells.


Figure 2: The apoptosis effect of supernova to E. coli cells. Light exposure greatly reduced the CFU number of bacteria cells, while darkness exposure allowed normal growth during a 48-h period of time. LB plates were grown for 24 h after light irradation/kept in darkenss.


Improvement

This part is an improved part of supernova (BBa_K1491017). The improvement was made by inserting supernova gene into a light-regulated expression system, lightoff (BBa_K2244009), cell apoptosis can thus be induced by firstly expressing supernova gene in darkness, and then expose cells to light illumination that results to ROS release and the subsequent cell death. The entire process is purely regulated by light. We believe this has improved the original use of supernova in a T7 system using IPTG induction, which requires additional chemical inducer.This improvement has made the use of supernova suicide system more convenient and more easily manipulated.


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
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 1045
    Illegal NheI site found at 1068
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 213
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1783
  • 1000
    COMPATIBLE WITH RFC[1000]