Difference between revisions of "Part:BBa K1933001"

Revision as of 09:29, 15 October 2016

INPNC fused to 6xHis tag

INPNC codes for N- and C- terminal domain of Ice-Nucleation Protein (INP) from Pseudomonas syringae , connected by a 6xHis tag to be easily identified by Western blotting[1]. This is used as an anchoring motif for the cell surface display of passengers. This domain does not have signal peptide for anchoring on the cell surface[2]. Surface displayed passengers retain enzyme activity even when it is linked to INPNC with 6xHis tag.
For more information, please visit [http://2016.igem.org/Team:Kyoto our wiki]

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BamHI site found at 952
23
COMPATIBLE WITH RFC[23]
25
INCOMPATIBLE WITH RFC[25]
Illegal NgoMIV site found at 72
Illegal NgoMIV site found at 405
Illegal AgeI site found at 823
1000
COMPATIBLE WITH RFC[1000]

Usage and Biology

We used this part for construction of BBa_K1933100, BBa_K1933101, BBa_K1933200

Characterization

Western blotting

Figure 1 : (a) 1. size marker, 2.Negative Control(BBa_K165002), 3.BclA-His-scFv (33kDa), 4.INPNC-His-scFv（63kDa）, 5.BclA-His-CBDcex(17kDa), 6.INPNC-His-CBDcex(47kDa), 7.LPP-OmpA-scFv-His(43kDa) (b) membrane fraction Western blotting against His tag. 1. marker, 2. Negative Control (BBa_K165002), 3. BclA-His-scFv (33kDa), 4. INPNC-His-scFv (63kDa), 5. BclA-His-CBDcex (17kDa), 6. INPNC-His-CBDcex (47kDa),7. Positive control:LPP-OmpA-scFv-His (43kDa)

Contribution

We improved BBa_K811005 from Penn 2012.

Reference

[1]Yim, Sung-Kun, et al. "Surface display of heme-and diflavin-containing cytochrome P450 BM3 in Escherichia coli: a whole cell biocatalyst for oxidation." Journal of microbiology and biotechnology, 20.4, (2010): 712-717.

[2]Park, Tae Jung, et al. "Surface display of recombinant proteins on Escherichia coli by BclA exosporium of Bacillus anthracis." Microbial cell factories, 12.1, (2013): 1.

@@ Line 17: / Line 17: @@
 ==Characterization==
-'''Western blotting '''
+==='''Western blotting '''===
 <!--綺麗な画像が作れれば差し替え-->
 [[file:T--Kyoto--western_for_K1933001.jpeg|thumb|center|400px|''' Figure 1 ''': ''' (a) ''' 1. size marker, 2.Negative Control(BBa_K165002), 3.BclA-His-scFv (33kDa), 4.INPNC-His-scFv（63kDa）, 5.BclA-His-CBDcex(17kDa), 6.INPNC-His-CBDcex(47kDa), 7.LPP-OmpA-scFv-His(43kDa) ''' (b) ''' membrane fraction Western blotting against His tag. 1. marker, 2. Negative Control (BBa_K165002), 3. BclA-His-scFv (33kDa), 4. INPNC-His-scFv (63kDa), 5. BclA-His-CBDcex (17kDa), 6. INPNC-His-CBDcex (47kDa),7. Positive control:LPP-OmpA-scFv-His (43kDa)]]