Part:BBa_K2328049
HU + GLBP + Linker.a + smURFP III + Histag.a + pMB1
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 893
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 210
Illegal NgoMIV site found at 732
Illegal NgoMIV site found at 1172
Illegal NgoMIV site found at 1691
Illegal NgoMIV site found at 1768 - 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 396
Illegal BsaI.rc site found at 813
Illegal SapI site found at 2854
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 GNB/LNB substrate-binding membrane protein (GL-BP) is a membrane protein belonging to the ATP binding cassette (ABC) protein family, Which transports lacto-N-biose (i.e.,N-acetyl-3-O-([3-D-galactopyranosyl)D-glucosamine) and galacto-N-biose (i.e.,N-acetyl-3-O-([3D-galactopyranosyl)-D-galactosamine) of bifidobacterium. ABC proteins are important membrane proteins that actively transport specific substances on the cell membranes of all organisms using an energy called adenosine triphosphate (ATP), and various ABC proteins are present on the cell membranes. Therefore, if an appropriate promoter is used, GL-BP, which is an ABC protein, is ubiquitously expressed in bacteria belonging to the genus bifidobacterium, which have a cellular function for expressing GL-BP on the surface thereof.
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.a is used to separate smURFP from GLBP.
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. 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 GLBP 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.
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] Part:BBa_K2328010.
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