Part:BBa_K2328054
HU + Linker I + AcmA + Linker II + Linker.a + smURFP III + Histag.a + pMB1
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 969
Illegal NgoMIV site found at 1046 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 2132
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. 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 Acma.
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. In the 3’end of the smURFP we also 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_K2328006.
[5] 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.
| None |