Difference between revisions of "Part:BBa K4488027"

Revision as of 03:21, 12 October 2022

Neae-intimin Construct Displaying anti-sfGFP Nanobody H

Surface display of anti-sfGFP nanobody H using the bacterial protein Neae-intimin. The nanobody is a modification of PDB: 3OGO-H. 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 anti-sfGFP nanobody H binds to sfGFP through a GFP bindings assay. Therefore, nanobody H can be effectively surface displayed using Neae-intimin which provides preliminary evidence that other types of nanobodies can be displayed and later selected using the same 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.

The construct also demonstrated effective binding to fuGFP.

Figure 3: Nanobody-GFP Binding Assay to validate H as a positive control and Neae-Intimin as a negative control for the binding to sfGFP. Measurements were taken at 470nm. Each bar is the average of 5 replicates, with error bars representing the standard error. Measurements were taken for a concentrated and equimolar amounts of GFP added to the nanobodies. Adding concentrated amounts of purified sfGFP markedly increased the error. Bars not sharing letters are significantly different based on a tukeyHSD post-hoc contrast.

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 anti-sfGFP nanobody H binds to sfGFP through a GFP bindings assay. Therefore, nanobody H can be effectively surface displayed using Neae-intimin which provides preliminary evidence that other types of nanobodies can be displayed and later selected using the same 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.]]
 The construct also demonstrated effective binding to fuGFP.
-[[File: sfGFP Binding Assay(470nm) .png|centre|thumb| Figure 3: Nanobody-GFP Binding Assay to validate H as a positive control and Neae-Intimin as a negative control for the binding to sfGFP. Measurements were taken at 470nm. Each bar is the average of 5 replicates, with error bars representing the standard error. Measurements were taken for a concentrated and equimolar amounts of GFP added to the nanobodies. Adding concentrated amounts of purified sfGFP markedly increased the error. Bars not sharing letters are significantly different based on a tukeyHSD post-hoc contrast.]]
+[[File: sfGFP Binding Assay(470nm) .png|centre|thumb| 700px| Figure 3: Nanobody-GFP Binding Assay to validate H as a positive control and Neae-Intimin as a negative control for the binding to sfGFP. Measurements were taken at 470nm. Each bar is the average of 5 replicates, with error bars representing the standard error. Measurements were taken for a concentrated and equimolar amounts of GFP added to the nanobodies. Adding concentrated amounts of purified sfGFP markedly increased the error. Bars not sharing letters are significantly different based on a tukeyHSD post-hoc contrast.]]
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