Difference between revisions of "Part:BBa K4283010"

 
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The design of the outer membrane vesicle (OMV) protein outlined in figure 1 contains three major structural domains: vesicle surface protein C terminus domain ClyA, cancer cell antigen Adpgk domain, and mouse IgG Fc fragment for the purpose of immune-enhancing and presenting antigen.
 
The design of the outer membrane vesicle (OMV) protein outlined in figure 1 contains three major structural domains: vesicle surface protein C terminus domain ClyA, cancer cell antigen Adpgk domain, and mouse IgG Fc fragment for the purpose of immune-enhancing and presenting antigen.
 +
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<b>Figure 1 </b>
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[[File:fig 3010 1.png|400px|thumb|center|Design of OMV protein producing part]]
 +
 +
<p>The fusion protein include a HA (hemagglutinin) tag for Western Blotting and, an enhanced GPR reporter gene. The design of the redesigned plasmid is shown in figure 2.</p>
 +
 +
<b>Figure 2 </b>
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[[File:fig 3010 2.png|600px|thumb|center|OMV protein with HA tag and report gene and plasmid pET-28b ClyA-Adpgk-mFC-eGFP]]
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<p>E.coli BL21 DE3 were transformed with the redesigned pET28b vector and transformants were induced with 0mM - 2mM IPTG and screened for GFP expression.</p>
 +
 +
<b>Figure 3 </b>
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[[File:fig 3010 3.png|600px|thumb|center|Detection of OMV through eGFP (model result)]]
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 +
<p>After induction at 37℃ for 4h a significant fluorescence signal was detected and is show in figure 5. The optimum IPTG concentration for expression was determined to be in the range of 0.4mM to 0.6mM. Figure 4 displays images of E.coli transformants expression enhance GFP.</p>
 +
 +
<b>Figure 4 </b>
 +
[[File:fig 3010 4.png|600px|thumb|center|Enhanced GFP expression of E.coli under fluorescence microscope]]
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<p>The signal of GFP florescent protein is indicative of OMV protein expression. It was decided to perform a western blot to confirm the weak band noted in figure 3c.</p>
 +
 +
<b>Figure 7 </b>
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[[File:fig 3010 7.png|600px|thumb|center| Western blot result using anti-HA tag antibody, after 37℃ 4h with 0.5mM IPTG
 +
(loading 20ul of each sample]]
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 +
Figure 7 shows the western blot image confirming the whole system was working.
 +
More detailed information for this part: https://2022.igem.wiki/sz-shd/engineering
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 +
 +
 +
  
 
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===Usage and Biology===
 
===Usage and Biology===
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K4283010 SequenceAndFeatures</partinfo>
 
  
  

Revision as of 14:30, 12 October 2022


Tumor antigen attached outer membrance vessicle(OMV-ClyA)

Outer membrane vesicles (OMVs) naturally secreted by E coli can work as delivery vehicles to carry display antigenic proteins on their surfaces and across intestinal epithelial barrier to where they can activate immune cells.

The design of the outer membrane vesicle (OMV) protein outlined in figure 1 contains three major structural domains: vesicle surface protein C terminus domain ClyA, cancer cell antigen Adpgk domain, and mouse IgG Fc fragment for the purpose of immune-enhancing and presenting antigen.

Figure 1

Design of OMV protein producing part

The fusion protein include a HA (hemagglutinin) tag for Western Blotting and, an enhanced GPR reporter gene. The design of the redesigned plasmid is shown in figure 2.

Figure 2

OMV protein with HA tag and report gene and plasmid pET-28b ClyA-Adpgk-mFC-eGFP

E.coli BL21 DE3 were transformed with the redesigned pET28b vector and transformants were induced with 0mM - 2mM IPTG and screened for GFP expression.

Figure 3

Detection of OMV through eGFP (model result)

After induction at 37℃ for 4h a significant fluorescence signal was detected and is show in figure 5. The optimum IPTG concentration for expression was determined to be in the range of 0.4mM to 0.6mM. Figure 4 displays images of E.coli transformants expression enhance GFP.

Figure 4

Enhanced GFP expression of E.coli under fluorescence microscope

The signal of GFP florescent protein is indicative of OMV protein expression. It was decided to perform a western blot to confirm the weak band noted in figure 3c.

Figure 7

Western blot result using anti-HA tag antibody, after 37℃ 4h with 0.5mM IPTG (loading 20ul of each sample

Figure 7 shows the western blot image confirming the whole system was working. More detailed information for this part: https://2022.igem.wiki/sz-shd/engineering