Part:BBa_K2244010
ColE promoter +supernova gene +Lev1 gene
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.
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.
LEV1 repressor(BBa_K2244005) 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 .
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.
Supernova is a mutant of killer red (BBa_K1491017),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.
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.
Improvenent
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 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 function more convenient and more easily manipulated. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1045
Illegal NheI site found at 1068 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 213
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1783
- 1000COMPATIBLE WITH RFC[1000]
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