Difference between revisions of "Part:BBa K4197007"

Latest revision as of 18:38, 10 October 2022

OmpA_Ana o 3 fusion

OmpA_Ana o 3 fusion to display cashew allergen on the E. coli cell surface.

Introduction

This part is composed of the gene coding for the allergen of cashew Ana o 3 (NCBI: AAL91665.1). The cashew allergy prevalence is higher than 0.08% in the US countries (Van der Valk and al. 2014) and Ana o 3 binds specific antibodies of 100% of the patients with cashew allergy (Sato and al. 2019). Ana o 3 has already been expressed in E. coli and was able to bind the IgE of patient with cashew allergie (Robotham and al. 2005).Ana o 3 was merged to the membrane protein OmpA of E. coli (BBa_K1694002) to display Ana o 3 on the surface of E. coli . This lipoprotein is the most abundant in E. coli's membrane with 100,000 copies per cell (Ortiz-Suarez and al. 2016) and is often used to display protein on the surface of bacteria (Yang and al. 2016).

Construction

Ana o 3 gene ordered on IDT was amplified by PCR using the high fidelity Phusion polymerase with the primers IF3_allergen (gccgcaagctttaatgatggtgatggtgatggtgatg) F and IF4_Ana o 3 (cctgtattttcagagcatggcgaaatttcttttattattg). Expected size of the amplicon was 479 bp.

Amplification product sizes were checked on EtBr stained agarose gel (Figure 1).

Figure 1: Ana o 3 amplified fragment. Expected size of the amplicons was 479 bp. PCR amplicon sizes Ana o 3 were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).

The products matched expected sizes and amplicons were further purified from the gel. The Ana o 3 construction was inserted into pET-21 b (+)_OmpA linearized by In-Fusion.

In-Fusion assemby reaction was transformed into Stellar competent cells. Transformants were selected on LB-ampicillin plates. 15 transformants were screened by colony PCR with primer pairs flanking the insertion zone (primers used: screening_inserts-F: ggttatgctagttattgctcagc and screening_inserts-R: ccgaaacaagcgctcatgagc). 4 positive transformants were detected (Figure 2).

Figure 2: pET21 b (+)_OmpA_Ana o 3 construction fragments from colony PCR. PCR amplicon sizes of colonies with Ana o 3 plasmid were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).

These transformants (colonies 1, 3 and 9) had their plasmid extracted by Miniprep and digested by Eco-RI and Eco-RV (expected size of the fragments: 5052 bp and 3211 pb) to assess the assembly (Figure 3).

Figure 3: restriction profile of pET-21 b (+)_Ana o 3 final construction. Enzymes were EcoRI and EcoRV. Plasmids were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).

The correct restriction maps were observed and these clones were further validated by sequencing. The plasmid was named pET-21 b (+)_OmpA_Ana o 3.

The plasmid was finally used to transform E. coli Tuner cells to express the OmpA_Ana o 3 construction at the cell membrane.

Validation

The plasmid was eventually used to transform E. coli Tuner cells in order to express the OmpA_Ana o 3 construction at the cell membrane. The expression and display controls should have been conducted using anti-Ana o 3 antibodies to check wether the allergen displayed on the bacteria were able to link to their specific IgE. However, due to the high price of these IgE, the experiment was not performed.

References

More information about the project for which the part was created: DAISY (INSA-UPS 2022)

Other parts to display allergens:
- OmpA_Ara h 2
- OmpA_Der p 2
- OmpA_Gal d 2

Van der Valk, J. P. M., J. Dubois, A. E., Gerth van Wijk, R., Wichers, H. J., de Jong, N. W. (2014). Systematic review on cashew nut allergy. Allergy. 69(6), 692–698. doi:10.1111/all.12401

Sato, S., Movérare, R., Ohya, Y., Ito, K., Nagao, M., Borres, M. P., & Ebisawa, M. (2019). Ana o 3–specific IgE is a predictive marker for cashew oral food challenge failure. The Journal of Allergy and Clinical Immunology : In Practice, 7(8), 2909–2911.e4. https://doi.org/10.1016/j.jaip.2019.04.049

Robotham, J. M., Wang, F., Seamon, V., Teuber, S. S., Sathe, S. K., Sampson, H. A., Beyer, K., Seavy, M., & Roux, K. H. (2005). Ana o 3, an important cashew nut (Anacardium occidentale L.) allergen of the 2S albumin family. Journal of Allergy and Clinical Immunology, 115(6), 1284–1290.

Ortiz-Suarez, M. L., Samsudin, F., Piggot, T. J., Bond, P. J., & Khalid, S. (2016). Full-Length OmpA : Structure, Function, and Membrane Interactions Predicted by Molecular Dynamics Simulations. Biophysical Journal, 111(8), 1692–1702. https://doi.org/10.1016/j.bpj.2016.09.009

Yang, Chao; Zhao, Qiao; Liu, Zheng; Li, Qiyun; Qiao, Chuanling; Mulchandani, Ashok; et al. (2016): Cell Surface Display of Functional Macromolecule Fusions on Escherichia coli for Development of an Autofluorescent Whole-Cell Biocatalyst. ACS Publications. Journal contribution. https://doi.org/10.1021/es800441t.s001

Sequence and Features

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal EcoRI site found at 130
Illegal XbaI site found at 47
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 130
Illegal NheI site found at 92
Illegal NotI site found at 1086
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 130
Illegal BamHI site found at 124
Illegal XhoI site found at 1095
23
INCOMPATIBLE WITH RFC[23]
Illegal EcoRI site found at 130
Illegal XbaI site found at 47
25
INCOMPATIBLE WITH RFC[25]
Illegal EcoRI site found at 130
Illegal XbaI site found at 47
1000
INCOMPATIBLE WITH RFC[1000]
Illegal SapI.rc site found at 757

@@ Line 1: / Line 1: @@
 __NOTOC__
-<partinfo>BBa_K4197003 short</partinfo>
+<partinfo>BBa_K4197007 short</partinfo>
-Brick expressing Ana o 3 at the surface of E. coli cell sortable by FACS
+OmpA_Ana o 3 fusion to display cashew allergen on the <i>E. coli</i> cell surface.
 <html>
 <h2>Introduction</h2>
-<p>The part expressing the gene of cashew nut Ana o 3 (<a href="https://parts.igem.org/Part:BBa_K4197007">K4197007</a>) has been completed with the ihfb800-RFP construction
+<p>This part is composed of the gene coding for the allergen of cashew Ana o 3 (NCBI: <a "https://www.ncbi.nlm.nih.gov/protein/AAL91665.1/">AAL91665.1</a>). The cashew allergy prevalence is higher than 0.08% in the US countries (Van der Valk and al. 2014) and Ana o 3 binds specific antibodies of 100% of the patients with cashew allergy  (Sato and al. 2019). Ana o 3 has already been expressed in <i>E. coli </i>and was able to bind the IgE of patient with cashew allergie (Robotham and al. 2005).Ana o 3 was merged to the membrane protein OmpA of <i>E. coli</i> (<a href="https://parts.igem.org/Part:BBa_K1694002">BBa_K1694002</a>) to display Ana o 3 on the surface of <i>E. coli </i>. This lipoprotein is the most abundant in <i>E. coli</i>'s membrane with 100,000 copies per cell (Ortiz-Suarez and al. 2016) and is often used to display protein on the surface of bacteria (Yang and al. 2016).</p>
-(<a href="https://parts.igem.org/Part:BBa_K41970012">K41970012</a>) to express red
-fluorescence. This red fluorescence allows sorting of bacteria by FACS. </p>
 <h2>Construction</h2>
-<p>The ihfb800-mRFP1 construction was amplified by PCR with the high-fidelity Phusion polymerase using BFP/RFP IF+BglII R site R
+<p>Ana o 3 gene ordered on IDT was amplified by PCR using the high fidelity Phusion polymerase with the primers IF3_allergen (gccgcaagctttaatgatggtgatggtgatggtgatg) F and IF4_Ana o 3 (cctgtattttcagagcatggcgaaatttcttttattattg). Expected size of the amplicon was  479 bp.</p>
-(cgcgggatcgagatctatcacgaggcagaatttcagat) and BFP/RFP IF+BglII R site F (tagaggatcgagatctctgaaacagtgcaaagctaaccc) primers. The expected size of the amplicon is 1622
-bp. The fragment was inserted on the linearized plasmid pET21b(+) with Ana o 3 (<a href="https://parts.igem.org/Part:BBa_K4197007">K4197007</a>) by In-Fusion. </p>
+<p>Amplification product sizes were checked on EtBr stained agarose gel (Figure 1).</p>
-<p>The resulting products were transformed into Stellar cells and transformants were selected. Colonies were screened by PCR using screening_insert_R
-(ccgaaacaagcgctcatgagc) and screening_insert_F (ggttatgctagttattgctcagc) primers. The expected sizes of amplicons was 2944 bp with RFP and 1453 bp without RFP.</p>
 <div class="center">
          <div class="thumb tnone">
              <div class="thumbinner" style="width:50%;">
-                 <a href="https://static.igem.wiki/teams/4197/wiki/parts/raph/anao3-check.png" class="image">
+                 <a href="https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-fragment.png" class="image">
-                     <img alt="" src="https://static.igem.wiki/teams/4197/wiki/parts/raph/anao3-check.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
+                     <img alt="" src="https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-fragment.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
                      <div class="magnify">
-                         <a href="https://static.igem.wiki/teams/4197/wiki/parts/raph/anao3-check.png" class="internal" title="Enlarge"></a>
+                         <a href="https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-fragment.png" class="internal" title="Enlarge"></a>
                      </div>
-                     <b>Figure 1: </b> <b>Verification of the insertion of RFP fragment in Ana o 3 with gel.</b>
+                     <i><b>Figure 1: Ana o 3 amplified fragment. Expected size of the amplicons was 479 bp.</b> PCR amplicon sizes Ana o 3 were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).</i>
-					The PCR screening was checked with 0.6% agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the
-					NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel). Colony 8 has shown the
-					right size.
                  </div>
              </div>
          </div>
      </div>
+</div>
-<h2>Validation</h2>
-<p>Successful colonies were further tested by digestion with NotI which cut both in the plasmid and in the mRFP1 gene (Figure 2). Correct sizes were expected to be
-and 6791 bp for Ara h 2.</p>
+<p>The products matched expected sizes and amplicons were further purified from the gel. The Ana o 3 construction was inserted into pET-21 b (+)_OmpA linearized by In-Fusion.</p>
+<p>In-Fusion assemby reaction was transformed into Stellar competent cells. Transformants were selected on LB-ampicillin plates. 15 transformants were screened by colony PCR with primer pairs flanking the insertion zone (primers used: screening_inserts-F: ggttatgctagttattgctcagc and screening_inserts-R: ccgaaacaagcgctcatgagc). 4 positive transformants were detected (Figure 2).</p>
 <div class="center">
-    <div class="thumb tnone">
+        <div class="thumb tnone">
-        <div class="thumbinner" style="width:80%;">
+            <div class="thumbinner" style="width:50%;">
-            <a href="https://static.igem.wiki/teams/4197/wiki/parts/raph/anao3-dig.png" class="image">
+                <a href="https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-screening.png" class="image">
-                <img alt="" src="https://static.igem.wiki/teams/4197/wiki/parts/raph/anao3-dig.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
+                    <img alt="" src="https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-screening.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
-                <div class="magnify">
+                    <div class="magnify">
-                    <a href="https://static.igem.wiki/teams/4197/wiki/parts/raph/anao3-dig.png" class="internal" title="Enlarge"></a>
+                        <a href="https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-screening.png" class="internal" title="Enlarge"></a>
+                    </div>
+                    <i><b>Figure 2: pET21 b (+)_OmpA_Ana o 3 construction fragments from colony PCR.</b> PCR amplicon sizes of colonies with Ana o 3 plasmid were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).</i>
                  </div>
-                <b>Figure 2: </b> <b>Digestion by NotI of Ana o 3 with mRFP1 insertion.</b>
-                The digestion product was checked with 0.6% agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the
-				NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel). colonies 21 and 23 present
-				the correct size for Ana o 3.
              </div>
          </div>
@@ Line 58: / Line 60: @@
 </div>
-<p>This construction has not shown red fluorescence on the microscope. After sequencing, a mutation has been revealed on the mRFP sequence which does not allow us
-to continue further with this construction.</p>
+<p>These transformants (colonies 1, 3 and 9) had their plasmid extracted by Miniprep and digested by Eco-RI and Eco-RV (expected size of the fragments:  5052 bp and 3211 pb) to assess the assembly (Figure 3).</p>
+<div class="center">
+        <div class="thumb tnone">
+            <div class="thumbinner" style="width:50%;">
+                <a href="https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-digestion.png" class="image">
+                    <img alt="" src="https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-digestion.png" width="100%" height=auto class="thumbimage" /></a>                  <div class="thumbcaption">
+                    <div class="magnify">
+                        <a href="https://static.igem.wiki/teams/4197/wiki/results/collection-of-allergens/ana-o-3-digestion.png" class="internal" title="Enlarge"></a>
+                    </div>
+                    <i><b>Figure 3: restriction profile of pET-21 b (+)_Ana o 3 final construction. Enzymes were EcoRI and EcoRV.</b> Plasmids were checked with agarose electrophoresis gel and revealed with EtBr. A theoretical gel is presented with each gel and the NEB 1 kb DNA ladder is used for the experimental gels (note that a different ladder is presented on the theoretical gel).</i>
+                </div>
+            </div>
+        </div>
+    </div>
+</div>
+<p>The correct restriction maps were observed and these clones were further validated by sequencing. The plasmid was named <b>pET-21 b (+)_OmpA_Ana o 3</b>.</p>
+<p>The plasmid was finally used to transform <i>E. coli</i> Tuner cells to express the OmpA_Ana o 3 construction at the cell membrane.</p>
+<h2>Validation</h2>
+<p>The plasmid was eventually used to transform <i>E. coli</i> Tuner cells in order to express the OmpA_Ana o 3 construction at the cell membrane. The expression and display controls should have been conducted using anti-Ana o 3 antibodies to check wether the allergen displayed on the bacteria were able to link to their specific IgE. However, due to the high price of these  IgE, the experiment was not performed. </p>
 <h2>References</h2>
+<p>More information about the project for which the part was created:<a href="https://2022.igem.wiki/toulouse-insa-ups/index.html"> DAISY (INSA-UPS 2022)</a> </p>
+<p>Other parts to display allergens:<br>
+    - <a href="https://parts.igem.org/Part:BBa_K4197008"> OmpA_Ara h 2</a> <br>
+    - <a href="https://parts.igem.org/Part:BBa_K4197006"> OmpA_Der p 2</a> <br>
+    - <a href="https://parts.igem.org/Part:BBa_K4197009"> OmpA_Gal d 2</a> <br>
+ </p>
 <ol>
-	<li> <a href="https://parts.igem.org/Part:BBa_K4197007">K4197007</a> </li>
+    <i>
-	<li> <a href="https://parts.igem.org/Part:BBa_K4197012">K4197012</a> </li>
-	<li> <a href="https://2022.igem.wiki/toulouse-insa-ups/index.html"> DAISY (INSA-UPS 2022)</a> </li>
-</ol>
+    <li>Van der Valk, J. P. M., J. Dubois, A. E., Gerth van Wijk, R., Wichers, H. J., de Jong, N. W. (2014). Systematic review on cashew nut allergy. Allergy. 69(6), 692–698. doi:10.1111/all.12401 </li>
+    <li>Sato, S., Movérare, R., Ohya, Y., Ito, K., Nagao, M., Borres, M. P., & Ebisawa, M. (2019). Ana o 3–specific IgE is a predictive marker for cashew oral food challenge failure. The Journal of Allergy and Clinical Immunology : In Practice, 7(8), 2909–2911.e4. https://doi.org/10.1016/j.jaip.2019.04.049</li>
+    <li>Robotham, J. M., Wang, F., Seamon, V., Teuber, S. S., Sathe, S. K., Sampson, H. A., Beyer, K., Seavy, M., & Roux, K. H. (2005). Ana o 3, an important cashew nut (Anacardium occidentale L.) allergen of the 2S albumin family. Journal of Allergy and Clinical Immunology, 115(6), 1284–1290.</li>
+   <li>Ortiz-Suarez, M. L., Samsudin, F., Piggot, T. J., Bond, P. J., & Khalid, S. (2016). Full-Length OmpA : Structure, Function, and Membrane Interactions Predicted by Molecular Dynamics Simulations. Biophysical Journal, 111(8), 1692–1702. https://doi.org/10.1016/j.bpj.2016.09.009</li>
+<li>Yang, Chao; Zhao, Qiao; Liu, Zheng; Li, Qiyun; Qiao, Chuanling; Mulchandani, Ashok; et al. (2016): Cell Surface Display of Functional Macromolecule Fusions on Escherichia coli for Development of an Autofluorescent Whole-Cell Biocatalyst. ACS Publications. Journal contribution. https://doi.org/10.1021/es800441t.s001</li>
+</i>
+</ol>
 </html>
@@ Line 76: / Line 132: @@
 <!-- -->
 <span class='h3bb'>Sequence and Features</span>
-<partinfo>BBa_K4197003 SequenceAndFeatures</partinfo>
+<partinfo>BBa_K4197007 SequenceAndFeatures</partinfo>
 <!-- Uncomment this to enable Functional Parameter display
 ===Functional Parameters===
-<partinfo>BBa_K4197003 parameters</partinfo>
+<partinfo>BBa_K4197007 parameters</partinfo>
 <!-- -->