Difference between revisions of "Part:BBa K1933100"

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==Characterization==
 
==Characterization==
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===colony PCR===
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===RT-PCR===
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[[file:T--Kyoto--RT-CBDcex.001.jpeg|300px|thumb|right|'''Figure 1''':  ''' RT-PCR ''']]
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<div style="float:right;"><gallery width=''300px'' heights=''300px''>
 
<div style="float:right;"><gallery width=''300px'' heights=''300px''>
 
file:experiments fig_membrane wes cex.png|'''Figure 1''':  ''' membrane fraction Western blotting against His tag '''
 
file:experiments fig_membrane wes cex.png|'''Figure 1''':  ''' membrane fraction Western blotting against His tag '''
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<br> Lane M, molecular weight size markers; Lanes 1, E. coli which didn’t express His-tagged proteins. (BBa_E1010) We used this parts as a negative control; Lanes 2, 4, and 6, E. coli harboring INPNC-6His fusion proteins, in the order of INPNC-6His-CBDclos(~47 kDa), INPNC-6His-CBDcex(~47 kDa), and INPNC-6His-scFv(~63 kDa); Lanes 3, 5, and 7, E. coli harboring BclA-6His fusion proteins, in the order of BclA-6His-CBDclos(~17 kDa), BclA-6His-CBDcex(~17 kDa), and BclA-6His-scFv(~63 kDa); Lanes 8, and 9, E. coli harboring Lpp-OmpA-scFv-6His(~43 kDa). (BBa_875004) We used this parts as a positive control.; Lanes 1~7, outer membrane fraction.; Lane 8, and 9, total proteins.
 
<br> Lane M, molecular weight size markers; Lanes 1, E. coli which didn’t express His-tagged proteins. (BBa_E1010) We used this parts as a negative control; Lanes 2, 4, and 6, E. coli harboring INPNC-6His fusion proteins, in the order of INPNC-6His-CBDclos(~47 kDa), INPNC-6His-CBDcex(~47 kDa), and INPNC-6His-scFv(~63 kDa); Lanes 3, 5, and 7, E. coli harboring BclA-6His fusion proteins, in the order of BclA-6His-CBDclos(~17 kDa), BclA-6His-CBDcex(~17 kDa), and BclA-6His-scFv(~63 kDa); Lanes 8, and 9, E. coli harboring Lpp-OmpA-scFv-6His(~43 kDa). (BBa_875004) We used this parts as a positive control.; Lanes 1~7, outer membrane fraction.; Lane 8, and 9, total proteins.
  
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[[file:T--Kyoto--experiments fig_RT-PCR.png|300px|thumb|right|'''Figure 3''':  ''' RT-PCR ''']]
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'''Whole cell Western Blotting'''
 
'''Whole cell Western Blotting'''
 
Anti-6His western blotting analysis of E. coli displaying BclA or INPNC-6His-fused scFv, CBDcex, and CBDclos.
 
Anti-6His western blotting analysis of E. coli displaying BclA or INPNC-6His-fused scFv, CBDcex, and CBDclos.

Revision as of 09:54, 15 October 2016


constitutive expression of CBDcex fused to INPNC with 6xHis tag

CBDcex fused to INPNC with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be therapeutic solution against norovirus infections. This protein in particular is a cellulose binding protein(CBDcex) fused to surface expression anchoring domain(INPNC), connected by a 6xHis tag to be easily identified by Western blotting. For more information, please visit [http://2016.igem.org/Team:Kyoto our wiki].

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 1012
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 132
    Illegal NgoMIV site found at 465
    Illegal AgeI site found at 883
  • 1000
    COMPATIBLE WITH RFC[1000]


Usage

Characterization

colony PCR

RT-PCR

Figure 1: RT-PCR


  • Figure 1: membrane fraction Western blotting against His tag

  • Figure 2: whole cell Western blotting against His tag
    Lane M, molecular weight size markers; Lanes 1, E. coli doesn’t express histagged proteins. (BBa_K165002) We used this parts as a negative control; Lanes 2, 4, and 6, E. coli harboring BclA-6His fusion proteins, in the order of BclA-6His-CBDclos(~17 kDa), BclA-6His-CBDcex(~17 kDa), and BclA-6His-scFv(~33 kDa); Lanes 3, 5, and 7, E. coli harboring INPNC-6His fusion proteins, in the order of INPNC-6His-CBDclos(~47 kDa), INPNC-6His-CBDcex(~47 kDa), and INPNC-6His-scFv(~63 kDa); Lanes 8, E. coli harboring Lpp-OmpA-scFv-6His(~ 43kDa). (BBa_875004) We used this parts as a positive control.; Lanes 1~8, total proteins.


membrane fraction Western blotting
Anti-6His western blotting analysis of E. coli displaying BclA or INPNC-6xHis-fused scFv, CBDcex, and CBDclos.
Lane M, molecular weight size markers; Lanes 1, E. coli which didn’t express His-tagged proteins. (BBa_E1010) We used this parts as a negative control; Lanes 2, 4, and 6, E. coli harboring INPNC-6His fusion proteins, in the order of INPNC-6His-CBDclos(~47 kDa), INPNC-6His-CBDcex(~47 kDa), and INPNC-6His-scFv(~63 kDa); Lanes 3, 5, and 7, E. coli harboring BclA-6His fusion proteins, in the order of BclA-6His-CBDclos(~17 kDa), BclA-6His-CBDcex(~17 kDa), and BclA-6His-scFv(~63 kDa); Lanes 8, and 9, E. coli harboring Lpp-OmpA-scFv-6His(~43 kDa). (BBa_875004) We used this parts as a positive control.; Lanes 1~7, outer membrane fraction.; Lane 8, and 9, total proteins.


Whole cell Western Blotting Anti-6His western blotting analysis of E. coli displaying BclA or INPNC-6His-fused scFv, CBDcex, and CBDclos.

Contribution

We improved BBa_K811005 from Penn 2012.

Reference

Judging

We fulfilled criteria listed below with this part.

  • Validated Part/ Validated Contribution
  • [http://2016.igem.org/Team:Kyoto/HP/Gold Integrated Human Practice]
  • Improve a previous part or project
  • [http://2016.igem.org/Team:Kyoto/Proof Proof of concept]