Part:BBa_K2328058
HU + Linker I + nonsense sequence + Linker II +Linker.a + smURFP III + Histag.a + pMB1
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 309
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 131
Illegal XhoI site found at 125 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 566
Illegal NgoMIV site found at 643 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 194
Illegal SapI site found at 1729
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.
Besides, HU consists of a promoter and an RBS of the B.longum hup gene. pMB1 is essential for the shuttling of the plasmid from E.coli to Bifidobacterium, which was obtained from Jilin University. Linker II and Linker.a are used to separate smURFP from anchor protein. The meaningless sequence is 150 bp and has the restriction enzyme cutting sites of XhoI、BglII、NcoI、NheI.
Biology
In order to fluoresce, smURFP must be combined with biliverdin (BV) .We have two solutions to make in vivo imaging come true. The first one is co-expression system and the other one is surface display system. To construct the co-expression system, the gene of fluorescent protein---smURFP and the gene of the precursor of biliverdin---HO-1 should be connected to the same expression vector and then transferred to our target bacteria. The precursor of biliverdin will be transferred to biliverdin through a series of conversion, and then fluorescent protein will combine with biliverdin directly in our target bacteria and glow in the bacteria. To construct the surface display system, the gene of fluorescent protein---smURFP and the gene of the anchoring protein should be connected to the same expression vector. After the recombinant plasmid is transferred to the target bacteria, the fluorescent protein and anchoring protein will express at the same time and become fusion protein, and then the fluorescent protein will be carried to the cell surface by anchoring protein. With the added biliverdin, fluorescent protein will combine with biliverdin and glow on the cell surface.
Reference
[1] Rodriguez EA,Tran GN , Gross LA, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein .[J].NATURE METHODS,2016:763-769.
[2] Part:BBa_K1932001.
[3] Part:BBa_K1932000.
[4] 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.
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