Difference between revisions of "Part:BBa K2328049"

 
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<partinfo>BBa_K2328049 parameters</partinfo>
 
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===Usage===
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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.
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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===
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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 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 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===
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[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.
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[2] [[Part:BBa_K1932001]].
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[3] [[Part:BBa_K1932000]].
 +
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[4] [[Part:BBa_K2328010]].

Latest revision as of 01:50, 27 October 2017


HU + GLBP + Linker.a + smURFP III + Histag.a + pMB1

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 893
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE 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
  • 1000
    INCOMPATIBLE 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

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 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 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.