Part:BBa_K1933100

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]

Characterization

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))

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]