Composite

Part:BBa_K1933101

Designed by: Tomoki Uchino   Group: iGEM16_Kyoto   (2016-10-10)

constitutive expression of CBDclos fused to INPNC with 6xHis tag

CBDclos fused to INPNC with 6xHis tag is one of a series of surface expressing fusion proteins that make up biodevice that aims to be the therapeutic solution against norovirus infections. This protein in particular is a cellulose binding protein(CBDclos) fused to surface expression anchoring domain(INPNC), connected by a 6xHis tag to be easily identified by Western blotting.
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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

We succeeded in surface expressing anti-NoV scFv BBa_K1933002 antibody using INPNC. We aimed to orally administrate this protein expressing E. coli, however, we obtained an overwhelmingly negative result from surveys conducted when asked "Would you feel uncomfortable with oral administration of sterilized recombinant organisms?" This is why we also constructed a parts that surface expresses cellulose binding domains (CBD). With this part, we can "leash" E.coli to cellulose and make them swiftly exit human digestive systems. We selected CBDclos as CBD for this part.

Characterization

Sequence confirmed!

Western blotting

We used whole-cell Westernlotting with anti-His tag antibody(Fig.1(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.1(b)).

Fig.1: (a) whole cell Western blotting against His tag. (b) membrane fraction Western blotting against His tag.
Negative Control:BBa_K165002, BclA-His-CBDclos:17 kDa, INPNC-His-CBDclos:47 kDa, Positive control(Lpp-OmpA-scFv-His):43 kDa

A band corresponding to INPNC-His-CBDclos (17 kDa) was not observed in both whole cell and membrane fraction Western blotting (Fig.1(a),(b)), indicating that fusion protein was not expressed.

Judging

We fulfilled criteria listed below with this part.

[edit]
Categories
//binding/cellulose
//cds/membrane
Parameters
chassisE. coli