Part:BBa_K4375018
MiniNano for bacterial surface display
This construction consists of 4 described parts. These are: Codon optimalised INPNC for Surface Display (BBa_K4375007), flexible GS-linker (BBa_K4375010), miRFP670 nano (BBa_K4375008), J23101* (BBa_K4375003). They allow the bacterium to be expelled to the tumor surface, thus allowing its visualisation and colonisation on the tumour surface.
Usage and Biology
The MiniNano is a compact construct meant for extracellular protein (like tumor-specific receptor, antigen, etc.) detection via fluorescence. This protein has 3 domains. (1) INPNC surface display module (INPNC codes for N- and C- terminal domain of Ice Nucleation Protein (INP) from Pseudomonas syringe, and it is used for displaying proteins on bacteria's outer membrane (BBa_K4375007). It is linked to (by a flexible GS-linker, BBa_K4375010) (2) a small, near-infrared emitting fluorescent protein, miRFP670 nano (BBa_K4375008), which is connected to an anti-EGFR specific Nanobody, 7D12, via llama-derived IgA hinge region linker (BBa_K4375011). Moreover, it contains a Myc epitope tag, so the specific interaction can be detected with labelled antibodies. This complex detection device is expressed under the constitutive promoter J23101* (BBa_K4375003) The Nanobody in the construct can be easily swapped with other protein domains, as its sequence is framed by BamHI and SphI restriction sites
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 1055
Illegal PstI site found at 1313 - 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 29
Illegal PstI site found at 1055
Illegal PstI site found at 1313 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 1055
Illegal PstI site found at 1313 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 1055
Illegal PstI site found at 1313
Illegal NgoMIV site found at 118
Illegal NgoMIV site found at 451
Illegal AgeI site found at 869
Illegal AgeI site found at 1523
Illegal AgeI site found at 1655 - 1000COMPATIBLE WITH RFC[1000]
References
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https://2012.igem.org/Team:Penn/Team
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