Part:BBa_K2328057
HU + Linker I + AcmA + Linker II + Linker.a + smURFP III + Histag.a
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 468
Illegal PstI site found at 581 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 468
Illegal NheI site found at 750
Illegal PstI site found at 581 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 468
Illegal BglII site found at 131
Illegal XhoI site found at 125 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 468
Illegal PstI site found at 581 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 468
Illegal PstI site found at 581
Illegal NgoMIV site found at 1007
Illegal NgoMIV site found at 1084 - 1000COMPATIBLE WITH RFC[1000]
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.
Acma and smURFP are joined together, and the fragment is inserted into pMG36e to construct a new plasmid. When the plasmid is expressed, smURFP is anchored to the cell surface by Acma, thus expressing the shape.
Besides, HU consists of a promoter and an RBS of the B.longum hup gene. Linker II and Linker.a are used to separate smURFP from Acma.
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_K1932000.
[3] Part:BBa_K2328006.
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