Difference between revisions of "Part:BBa K2114002"

Revision as of 22:43, 18 October 2016

aGFPnano_HA_G4S_cotZ

N-terminal fusion of anti-GFP nanobody to spore crust gene cotZ by a flexible GGGGS linker.

Usage and Biology

Figure 1: Schematic representation of fusion protein.

This part includes the anti-GFP nanobody (describted by Kubala et al. [Ref]) fused by a flexible GGGGS linker [Ref] to the B. subtilis spore crust protein CotZ in order to be displayed on the spore surface. The hemagglutinin epitope tag was included in the fusion construct for convenient detection by specific anti-HA antibodies. The cotZ gene was amplified from the genome of B. subtilis and the anti-GFP nanobody was amplified from an expression plasmid. The HA tag and the linker were introduced by primer extensions. Both PCR fragments were assembled by Gibson cloning into pSB1C3. The fusion construct can released by XbaI and PstI and cloned alongside with an appropriate promoter into an integration vector for B. subtilis by 3A assembly. [Figure of construct: Representation of the fusion protein.]

Characterization

I) Expression: WB
II) Localizaion: FACS antiHA-Alexa647
III) Binding of GFP: FACS

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 447
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

@@ Line 6: / Line 6: @@
 ===Usage and Biology===
-This part includes the anti-GFP nanobody (describted by Kubala et al. [Ref]) fused by a flexible GGGGS linker [Ref] to the B. subtilis spore crust protein CotZ in order to be displayed on the spore surface. The hemagglutinin epitope tag was included in the fusion construct for convenient detection by specific anti-HA antibodies. The cotZ gene was amplified from the genome of B. subtilis and the anti-GFP nanobody was amplified from an expression plasmid. The HA tag and the linker were introduced by primer extensions. Both PCR fragments were assembled by Gibson cloning into pSB1C3. The fusion construct can released by XbaI and PstI and cloned alongside with an appropriate promoter into an integration vector for B. subtilis by 3A assembly. [Figure of construct: Representation of the fusion protein.]
+[[File:iG16_schematic_BBa_K2114002.png|350px|thumb|left|Figure 1: Schematic representation of fusion protein.]]
+This part includes the anti-GFP nanobody (describted by Kubala et al. '''[Ref]''') fused by a flexible GGGGS linker '''[Ref]''' to the B. subtilis spore crust protein CotZ in order to be displayed on the spore surface. The hemagglutinin epitope tag was included in the fusion construct for convenient detection by specific anti-HA antibodies. The cotZ gene was amplified from the genome of B. subtilis and the anti-GFP nanobody was amplified from an expression plasmid. The HA tag and the linker were introduced by primer extensions. Both PCR fragments were assembled by Gibson cloning into pSB1C3. The fusion construct can released by XbaI and PstI and cloned alongside with an appropriate promoter into an integration vector for B. subtilis by 3A assembly. [Figure of construct: Representation of the fusion protein.]