Difference between revisions of "Part:BBa K1933100"

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[[file:T--Kyoto--Western blotting Whole cell and membrane fraction.jpeg|600px|thumb|center|'''Fig.2''':  ''' (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)]]
 
[[file:T--Kyoto--Western blotting Whole cell and membrane fraction.jpeg|600px|thumb|center|'''Fig.2''':  ''' (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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A band corresponding to INPNC-His-CBDcex (47kDa) was observed in both of whole cell and membrane fraction Western blotting(Fig.2(a),(b))
  
 
==Reference==
 
==Reference==

Revision as of 18:33, 16 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

We designed PCR primers so that we would obtain PCR product with the length of 300 bp after RT-PCR. For negative control, reverse transcription was not performed.

Fig.1: RT-PCR
1. marker, 2. INP-His-CBDcex (negative control), 3. INP-His-CBDcex

A band corresponding to 300 bp was observed only in INP-His-scFv sample, not in negative control(Fig.1). This result suggests that INP-His-CBDcex was successfully transcribed in E.coli.

Western blotting

Fig.2: (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)

A band corresponding to INPNC-His-CBDcex (47kDa) was observed in both of whole cell and membrane fraction Western blotting(Fig.2(a),(b))

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]