Difference between revisions of "Part:BBa K1933100"

Revision as of 06:06, 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

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

Western blotting

Figure 1: (a) whole cell Western blotting against His tag. (b) membrane fraction Western blotting against His tag.

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]

@@ Line 25: / Line 25: @@
 [[file:T--Kyoto--Western blotting Whole cell and membrane fraction.jpeg|600px|thumb|center|'''Figure 1''':  ''' (a) '''whole cell Western blotting against His tag. ''' (b) '''membrane fraction Western blotting against His tag. ''']]
-[[file:experiments fig_membrane wes cex.png|200px|thumb|center|'''Figure 1''':  ''' membrane fraction Western blotting against His tag ''']]
-[[file:T--Kyoto--whole cell Wb.PNG|200px|thumb|center|'''Figure 2''':  ''' whole cell Western blotting against His tag '''<br> 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.]]
-<br>''' membrane fraction Western blotting'''
-<br>Anti-6His western blotting analysis of E. coli displaying BclA or INPNC-6xHis-fused scFv, CBDcex, and CBDclos.
-<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.
-'''Whole cell Western Blotting'''
-Anti-6His western blotting analysis of E. coli displaying BclA or INPNC-6His-fused scFv, CBDcex, and CBDclos.
 ==Reference==