Part:BBa_K2328060
nonsense sequence + Linker II + Linker.a + smURFP III + Histag.a
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 165
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 422
Illegal NgoMIV site found at 499 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 50
Usage
smURFP (small ultra-red FP) is the most important part in our group. It is desirable for our in-vivo imaging because with it molecule less light is scattered, absorbed, or re-emitted by endogenous biomolecules compared with cyan, green, yellow and orange FPs. smURFP can covalently attaches a biliverdin (BV) chromophore without a lyase, and has 642/670 nm excitation - emission peaks, a large extinction coefficient and quantum yield, and photostability comparable to that of eGFP. In order to make it expressed in bifidobacterium longum, we have made this sequence optimized. The meaningless sequence is 150 bp and has the restriction enzyme cutting sites of XhoI、BglII、NcoI、NheI. Linker II and Linker.a are used to separate smURFP from other core parts.
Biology
Bifidobacterium longum is a strictly anaerobic bacterium and there’s no oxygen in anaerobic bacteria, so the co- expression system won’t work, we choose the system of surface display. Our concept is proposed that the smURFP should be fused with an anchor sequence and displayed on the surface of a microbial cell so that they can be combined with BV. In our constructed plasmid the anchor sequence and our target protein smURFP is linked from the 5’ end side. In the 3’end of the smURFP we added his-tag so that we can testify whether the smURFP is expressed or not by using confocal. In order to add different anchor sequences more conveniently, we use the meaningless sequence with four restriction enzyme cutting sites on it as an instead.
Reference
[1] Yamamoto S, Wada J, Katayama T, Jikimoto T, Nakamura M, Kinoshita S, et al. (2010) Genetically modified Bifidobacterium displaying Salmonella-antigen protects mice from lethal challenge of Salmonella Typhimurium in a murine typhoid fever model. Vaccine 28: 6684–6691.
[2] Rodriguez EA,Tran GN , Gross LA, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein .[J].NATURE METHODS,2016:763-769.
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