Difference between revisions of "Part:BBa K1933001"

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==Characterization==
 
==Characterization==
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We ''' improved '''<big> [https://parts.igem.org/Part:BBa_K811005 BBa_K811005]</big> from Penn 2012!!
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====We ''' improved '''<big> [https://parts.igem.org/Part:BBa_K811005 BBa_K811005]</big> from Penn 2012!!====
  
 
===Western blotting===
 
===Western blotting===

Revision as of 06:00, 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.
1. size marker, 2.Negative Control(BBa_K165002), 3.BclA-His-scFv (33kDa), 4.INPNC-His-scFv（63kDa）, 5.BclA-His-CBDcex(17kDa), 6.INPNC-His-CBDcex(47kDa), 7.LPP-OmpA-scFv-His(43kDa)
(b) membrane fraction Western blotting against His tag.
1. marker, 2. Negative Control (BBa_K165002), 3. BclA-His-scFv (33kDa), 4. INPNC-His-scFv (63kDa), 5. BclA-His-CBDcex (17kDa), 6. INPNC-His-CBDcex (47kDa),7. Positive control:LPP-OmpA-scFv-His (43kDa)



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