Difference between revisions of "Part:BBa K1933001"

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===Western blotting===
 
===Western blotting===
 
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[[file:T--Kyoto--Western blotting Whole cell and membrane fraction.jpeg|600px|thumb|center|'''Figure 1''':  ''' (a) '''whole cell Western blotting against His tag. ''' (b) '''membrane fraction Western blotting against His tag.<br> Negative Control: BBa_K165002, BclA-His-scFv: 33 kDa, INPNC-His-scFv: 63 kDa, BclA-His-CBDcex: 17 kDa, INPNC-His-CBDcex: 47 kDa ,Positive control:LPP-OmpA-scFv-His (43 kDa)]]
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[[file:T--Kyoto--Western blotting Whole cell and membrane fraction.jpeg|600px|thumb|center|'''Figure 1''':  ''' (a) '''whole cell Western blotting against His tag. ''' (b) '''membrane fraction Western blotting against His tag.<br> Negative Control:BBa_K165002, BclA-His-scFv:33 kDa, INPNC-His-scFv:63 kDa, BclA-His-CBDcex:17 kDa, INPNC-His-CBDcex:47 kDa, Positive control:LPP-OmpA-scFv-His (43 kDa)]]
  
 
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Revision as of 06:16, 16 October 2016


INPNC fused to 6xHis tag

INPNC codes for N- and C- terminal domain of Ice-Nucleation Protein (INP) from Pseudomonas syringae , connected by a 6xHis tag to be easily identified by Western blotting[1]. This is used as an anchoring motif for the cell surface display of passengers. This domain does not have signal peptide for anchoring on the cell surface[2]. Surface displayed passengers retain enzyme activity even when it is linked to INPNC with 6xHis tag.
For more information, please visit [http://2016.igem.org/Team:Kyoto our wiki]


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 952
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 72
    Illegal NgoMIV site found at 405
    Illegal AgeI site found at 823
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage and Biology


We used this part for construction of BBa_K1933100, BBa_K1933101, BBa_K1933200

Characterization

We improved BBa_K811005 from Penn 2012!!

Western blotting

Figure 1: (a) whole cell Western blotting against His tag. (b) membrane fraction Western blotting against His tag.
Negative Control:BBa_K165002, BclA-His-scFv:33 kDa, INPNC-His-scFv:63 kDa, BclA-His-CBDcex:17 kDa, INPNC-His-CBDcex:47 kDa, Positive control:LPP-OmpA-scFv-His (43 kDa)




Please see 　BBa_K1933100, BBa_K1933101, and BBa_K1933200 for full characterization .

Contribution

Reference

[1]Yim, Sung-Kun, et al. "Surface display of heme-and diflavin-containing cytochrome P450 BM3 in Escherichia coli: a whole cell biocatalyst for oxidation." Journal of microbiology and biotechnology, 20.4, (2010): 712-717.


[2]Park, Tae Jung, et al. "Surface display of recombinant proteins on Escherichia coli by BclA exosporium of Bacillus anthracis." Microbial cell factories, 12.1, (2013): 1.

Judging