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.

Western blotting

We used whole-cell Westernlotting with anti-His tag antibody(Fig.2(a)). Then, we prepared the membrane fraction from the E. coli lysate. Membrane fraction was solubilized and used for Nickel Sepharose purification and precipitates were examined by Western blotting against His tag(Fig.2(b)).

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 whole cell and membrane fraction Western blotting (Fig.2(a),(b)), which confirms expression of the fusion proteins, and suggests that CBDcex was surface expressed as expected.

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]