Difference between revisions of "Part:BBa K2328061"

Latest revision as of 01:27, 27 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. HU consists of a promoter and an RBS of the B.longum hup gene. The meaningless sequence is 150 bp and has the restriction enzyme cutting sites of XhoI、BglII、NcoI、NheI. Linker I, II and Linker.a are used to separate smURFP from other core parts.

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.

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.

@@ Line 18: / Line 18: @@
 ===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 150 bp and has the restriction enzyme cutting sites of XhoI、BglII、NcoI、NheI.
+HU consists of a promoter and an RBS of the B.longum hup gene. The meaningless sequence is 150 bp and has the restriction enzyme cutting sites of XhoI、BglII、NcoI、NheI. Linker I, II and Linker.a are used to separate smURFP from other core parts.
 ===Biology===