Difference between revisions of "Part:BBa K2328052"
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===Biology===
 
===Biology===
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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.
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Bifidobacterium longum is an strictly anaerobic bacteria 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 glbp 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 are 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.
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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 GL-BP and displayed on the surface of a microbial cell so that they can be combined with BV.
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===Reference===
 
===Reference===

Revision as of 17:20, 26 October 2017


GLBP + Linker.a + smURFP III + Histag.a

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 771
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 88
    Illegal NgoMIV site found at 610
    Illegal NgoMIV site found at 1050
    Illegal NgoMIV site found at 1569
    Illegal NgoMIV site found at 1646
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 274
    Illegal BsaI.rc site found at 691


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, linker.a is used to separate smURFP from GLBP.

Biology

Bifidobacterium longum is an strictly anaerobic bacteria 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 glbp 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 are 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_K2328010.