Difference between revisions of "Part:BBa K2332025"
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As a more immediate induction of cell aggregation, we aimed to develop a post-translational light switch in which cells are constitutively expressing either Intimin’ fusion proteins with SpyTag or an inactive version of SpyCatcher. Only upon light exposure, SpyCatcher is able to bind SpyTag resulting in a much faster response than transcriptional induction. This is achieved by incorporating a photocaged unnatural amino acid (UAA), Ne-methyl-L-lysine, in place of the reactive lysine in SpyCatcher required for the covalent bond formation with the SpyTag aspartate residue. Upon exposure to UV light (20min, 365nm), the “cage” group in the unnatural photocaged amino acid is cleaved off revealing the native amino acid and a biologically active protein. This approach also adds a layer of bio-containment as cells will only function when externally supplied with UAA.
 
As a more immediate induction of cell aggregation, we aimed to develop a post-translational light switch in which cells are constitutively expressing either Intimin’ fusion proteins with SpyTag or an inactive version of SpyCatcher. Only upon light exposure, SpyCatcher is able to bind SpyTag resulting in a much faster response than transcriptional induction. This is achieved by incorporating a photocaged unnatural amino acid (UAA), Ne-methyl-L-lysine, in place of the reactive lysine in SpyCatcher required for the covalent bond formation with the SpyTag aspartate residue. Upon exposure to UV light (20min, 365nm), the “cage” group in the unnatural photocaged amino acid is cleaved off revealing the native amino acid and a biologically active protein. This approach also adds a layer of bio-containment as cells will only function when externally supplied with UAA.
  
 
To achieve this, we introduced an amber stop codon (TAG) in place of the reactive Lys 31 residue (Lys31X) in SpyCatcher. Amberless E. coli cells also have to express pyrrolysyl tRNA (pyIT/tRNAPylCUA) from M. mazei and pyrrolysyl-tRNA synthetase from M. barkeri and the UAA must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognized by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain. The orthogonality of this system has shown to work in both E. coli and mammalian cells.  
 
To achieve this, we introduced an amber stop codon (TAG) in place of the reactive Lys 31 residue (Lys31X) in SpyCatcher. Amberless E. coli cells also have to express pyrrolysyl tRNA (pyIT/tRNAPylCUA) from M. mazei and pyrrolysyl-tRNA synthetase from M. barkeri and the UAA must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognized by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain. The orthogonality of this system has shown to work in both E. coli and mammalian cells.  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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===Functional Parameters===
 
===Functional Parameters===
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Revision as of 19:08, 20 October 2017


Intimin'-mutSpyCatcher (Lys31X, for photocaging)

As a more immediate induction of cell aggregation, we aimed to develop a post-translational light switch in which cells are constitutively expressing either Intimin’ fusion proteins with SpyTag or an inactive version of SpyCatcher. Only upon light exposure, SpyCatcher is able to bind SpyTag resulting in a much faster response than transcriptional induction. This is achieved by incorporating a photocaged unnatural amino acid (UAA), Ne-methyl-L-lysine, in place of the reactive lysine in SpyCatcher required for the covalent bond formation with the SpyTag aspartate residue. Upon exposure to UV light (20min, 365nm), the “cage” group in the unnatural photocaged amino acid is cleaved off revealing the native amino acid and a biologically active protein. This approach also adds a layer of bio-containment as cells will only function when externally supplied with UAA.

To achieve this, we introduced an amber stop codon (TAG) in place of the reactive Lys 31 residue (Lys31X) in SpyCatcher. Amberless E. coli cells also have to express pyrrolysyl tRNA (pyIT/tRNAPylCUA) from M. mazei and pyrrolysyl-tRNA synthetase from M. barkeri and the UAA must be supplemented in the media. The pyrrolysyl-tRNA synthetase catalyses the acylation of the suppressor tRNACUA with the UAA. During translation, the UAG amber codon in the mRNA is recognized by the acylated tRNACUA and the UAA will be added to the growing polypeptide chain. The orthogonality of this system has shown to work in both E. coli and mammalian cells.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 880
    Illegal NgoMIV site found at 1621
  • 1000
    COMPATIBLE WITH RFC[1000]