Part:BBa_K2328040
HU + smURFP III + Histag.a + RBS III + HO-1 III + pMB1
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 840
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 840
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 840
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 840
Illegal NgoMIV site found at 330
Illegal NgoMIV site found at 407 - 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 2256
Usage
It’s a co-expression system. smURFP (small ultra-red FP) is an 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. HO-1 is the gene of the precursor of biliverdin. HO-1 can use the materials of the E.coil to produce biliverdin. So we want to construct a plasmid which can both express the smURFP gene and HO1 gene. Through this contruction, we can achieve the co-expression in the E.coil. Both the smURFP and biliverdin are produced by E.coil, so they can connect directly within the E.coil to produce fluorescence under the wavelength of 642nm without to add BV additionally. Besides, smURFP III and HO-1 III are both the codon-optimized version for higher expresion in Bifidobacterium longum. RBS III is used to construct the co-expression structure of smURFP and HO-1 linked behind RBS. 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.
Biology
In order to produce fluoresce, smURFP must be combined with biliverdin (BV) .So one of our method is co-expression. Because the HO-1 needs to use oxygen to produce BV, it is adoptable in E.coil which is a kind of facultative anaerobic bacteria. And the HO-1 gene is from the Block Library. Through this contruction, we can achieve the co-expression in the E.coil. Both the smURFP and biliverdin are produced by E.coil, so they can connect directly within the E.coil to produce fluorescence under the wavelength of 642 nm. 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] Dong Chen, Jason D Brown, Yukie Kawasaki, Jerry Bommer and Jon Y Takemoto. Scalable production of biliverdin IXα by Escherichia coli. [J].BMC Biotechnology, 2012.
[3] Part:BBa_K1932001.
[4] Part:BBa_K1932000.
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