Difference between revisions of "Part:BBa K4488029"

Revision as of 03:23, 12 October 2022

Neae-intimin Construct Displaying anti-sfGFP Nanobody 6

Surface display of anti-sfGFP nanobody 6 using the bacterial protein Neae-intimin. The nanobody is a modification of GenBank ID: AIF28006. There is a linker between the Neae-intimin gene and the nanobody sequence. It has been optimised to express in pUS250 TOP10 E.coli.

Usage and Biology

Our project demonstrated that other anti-sfGFP nanobodies binds to sfGFP through a GFP bindings assay. While nanobody 6 does not effectively surface displayed using Neae-intimin, we still provided preliminary evidence that other types of nanobodies can be displayed and later selected using a similar construct.

Figure 1: Binding of anti-GFP nanobodies H, 2, 3, 6 and 7 to sfGFP, fuGFP and eGFP, measured on the plate reader at 400nm (full settings can be found here). Measurements at 400nm are taken to optimise for fuGFP fluorescence. This graph shows no significant binding to fuGFP when compared to the Neae negative control.

Figure 2: Binding of anti-GFP nanobodies H, 2, 3, 6 and 7 to sfGFP, fuGFP and eGFP, measured on the plate reader at 470nm (full settings can be found here). This graph shows significant binding to sfGFP for nanobodies H, 2, 3 and 7 when compared to Neae negative control. Nanobody 6 does not appear to bind sfGFP more than the negative control. Nanobody H appears to bind the most to sfGFP.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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 Our project demonstrated that other anti-sfGFP nanobodies binds to sfGFP through a GFP bindings assay. While nanobody 6 does not effectively surface displayed using Neae-intimin, we still provided preliminary evidence that other types of nanobodies can be displayed and later selected using a similar construct.
-[[File: Binding of anti-fuGFP Nanobodies to GFPs (400nm) .png|centre|thumb| Figure 1: Binding of anti-GFP nanobodies H, 2, 3, 6 and 7 to sfGFP, fuGFP and eGFP, measured on the plate reader at 400nm (full settings can be found <span class="plainlinks">[https://2022.igem.wiki/sydney-australia/protocols here]</span>). Measurements at 400nm are taken to optimise for fuGFP fluorescence. This graph shows no significant binding to fuGFP when compared to the Neae negative control.]]
+[[File: Binding of anti-fuGFP Nanobodies to GFPs (400nm) .png|centre|thumb|700px| Figure 1: Binding of anti-GFP nanobodies H, 2, 3, 6 and 7 to sfGFP, fuGFP and eGFP, measured on the plate reader at 400nm (full settings can be found <span class="plainlinks">[https://2022.igem.wiki/sydney-australia/protocols here]</span>). Measurements at 400nm are taken to optimise for fuGFP fluorescence. This graph shows no significant binding to fuGFP when compared to the Neae negative control.]]
-[[File: Binding of anti-fuGFP Nanobodies to GFPs (470nm) .png|centre|thumb| Figure 2: Binding of anti-GFP nanobodies H, 2, 3, 6 and 7 to sfGFP, fuGFP and eGFP, measured on the plate reader at 470nm (full settings can be found  <span class="plainlinks">[https://2022.igem.wiki/sydney-australia/protocols here]</span>). This graph shows significant binding to sfGFP for nanobodies H, 2, 3 and 7 when compared to Neae negative control. Nanobody 6 does not appear to bind sfGFP more than the negative control. Nanobody H appears to bind the most to sfGFP.]]
+[[File: Binding of anti-fuGFP Nanobodies to GFPs (470nm) .png|centre|thumb| 700px|Figure 2: Binding of anti-GFP nanobodies H, 2, 3, 6 and 7 to sfGFP, fuGFP and eGFP, measured on the plate reader at 470nm (full settings can be found  <span class="plainlinks">[https://2022.igem.wiki/sydney-australia/protocols here]</span>). This graph shows significant binding to sfGFP for nanobodies H, 2, 3 and 7 when compared to Neae negative control. Nanobody 6 does not appear to bind sfGFP more than the negative control. Nanobody H appears to bind the most to sfGFP.]]
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