Part:BBa_K2332040
Blue light inducible expression of Intimin'-GFP-SpyTag (Pblind Intimin'-GFP-SpyTag)
This construct displays SpyTag fused to GFP on the cell surface of E. coli cells under the control of Pblind, a blue light inducible promoter. Cells must also express EL222 (BBa_K2332004). Upon blue-light (465nm) exposure, EL222 dimerises and binds to its DNA-binding region within Pblind to recruit RNAP and induce transcription of Intimin'-GFP-SpyTag.
Intimin' is a truncated version of Intimin (EaeA intimin), an outer membrane surface protein, which has shown to display proteins of similar size to GFP fused to its N terminal. This construct allows to directly observe through fluorescence microscopy the functionality of Intimin for cell surface display under blue light induction.
This construct can be used in combination with SpyCatcher constructs. SpyTag (13 amino acids) and SpyCatcher (138 amino acids, 15 kDa) are protein binding partners that form spontaneous irreversible isopeptide bonds between an aspartate residue in SpyTag and a lysine residue in SpyCatcher. These binding partners originate from CnaB2 (immunoglobulin-llike collagen adhesin domain) of the FbaB protein, found in the invasive strains of S. pyogenes. Thus, this construct allows to directly observe through fluorescence microscopy SpyTag localization on the cell surface, and most importantly its co-localization with SpyCatcher variants. Particularly for our project, it would allow us to directly observe cell-cell binding post blue-light exposure when used in combination with cells expressing intimin'-SpyCatcher.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal SpeI site found at 2844
- 12INCOMPATIBLE WITH RFC[12]Illegal SpeI site found at 2844
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal SpeI site found at 2844
- 25INCOMPATIBLE WITH RFC[25]Illegal SpeI site found at 2844
Illegal NgoMIV site found at 880
Illegal NgoMIV site found at 1621 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 2715
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