Difference between revisions of "Part:BBa K2328061"

 
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K2328061 short</partinfo>
 
<partinfo>BBa_K2328061 short</partinfo>
  
−
arashi
+
The original part [[Part:BBa_I15008]] has a barcode after coding sequence. So we submit a new part without the barcode for our composite parts. But we will upload all the data and infomation in both pages.
  
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 17: Line 16:
 
<partinfo>BBa_K2328061 parameters</partinfo>
 
<partinfo>BBa_K2328061 parameters</partinfo>
 
<!-- -->
 
<!-- -->
 +
===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 meaningless sequence is 150bp and have the restriction enzyme cutting sites of XhoI、BglII、NcoI、NheI
 +
li.
 +
 +
===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 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 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.In order to add different anchor sequences more conveniently, we use the meaningless sequence with four restriction enzyme cutting sites on it as a instead.
 +
 +
===Reference===
 +
[1]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.
 +
[2]Rodriguez EA,Tran GN , Gross LA, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein .[J].NATURE METHODS,2016:763-769

Revision as of 13:43, 26 October 2017

HU + Linker I + nonsense sequence + Linker II + Linker.a + smURFP III + Histag.a

The original part Part:BBa_I15008 has a barcode after coding sequence. So we submit a new part without the barcode for our composite parts. But we will upload all the data and infomation in both pages.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 309
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 131
    Illegal XhoI site found at 125
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 566
    Illegal NgoMIV site found at 643
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI site found at 194


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 meaningless sequence is 150bp and have the restriction enzyme cutting sites of XhoI、BglII、NcoI、NheI li.

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 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 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.In order to add different anchor sequences more conveniently, we use the meaningless sequence with four restriction enzyme cutting sites on it as a instead.

Reference

[1]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. [2]Rodriguez EA,Tran GN , Gross LA, et al. A far-red fluorescent protein evolved from a cyanobacterial phycobiliprotein .[J].NATURE METHODS,2016:763-769