Difference between revisions of "Part:BBa K3809010"
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To verify that ESR1 would fold correctly and there would be no steric obstacle, we simulated the protein with its corresponding additions using the tool Robetta (https://robetta.bakerlab.org/). When we recieved the model, we made an structural alignment using the natural protein as reference with a PDB code 2IOG (Protein Data Bank, rcsb.org). Therefore, we concluded that the addition of the Linker, His Tag and Signal Peptide were not interfering with the correct folding of ESR1. Furthermore, we proved that the His Tag and the linker were exposed, so we could use them for the purposed we established previously.  
 
To verify that ESR1 would fold correctly and there would be no steric obstacle, we simulated the protein with its corresponding additions using the tool Robetta (https://robetta.bakerlab.org/). When we recieved the model, we made an structural alignment using the natural protein as reference with a PDB code 2IOG (Protein Data Bank, rcsb.org). Therefore, we concluded that the addition of the Linker, His Tag and Signal Peptide were not interfering with the correct folding of ESR1. Furthermore, we proved that the His Tag and the linker were exposed, so we could use them for the purposed we established previously.  
  
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<p class=MsoNormal><b><span lang=EN-US style='font-size:10.0pt;line-height:
 
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color:black;mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></b></p>
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color:black;mso-ansi-language:EN-US'></span></b></p>
  
 
<p class=MsoNormal><b><span lang=EN-US style='font-size:10.0pt;line-height:
 
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color:black;mso-ansi-language:EN-US'>Biology and characterization of
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color:black;mso-ansi-language:EN-US'>Characterization of BBa_K3809012</span></b></p>
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BBa_K3809010<o:p></o:p></span></b></p>
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<p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;line-height:107%;
 
<p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;line-height:107%;
 
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font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";
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color:black;mso-ansi-language:EN-US'>We wanted to test if our protein receptor
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color:black;mso-ansi-language:EN-US'>For the characterization of the protein
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would fold adequately. However, before we could enter the lab, we could not do
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BBa_K380912 we transformed the plasmid in <i>E. coli</i> BL21. We then examined
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it experimentally, so we recurred to an <span class=GramE><i>in silico</i></span>
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the growth of different colonies and performed a Colony PCR to determine if
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analysis. We wanted to verify that all the modifications and features we added wouldn’t
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they had the insert or not. The result can be seen in Figure 1. And 2. <span
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interfere with the correct folding of BBa_K3809010; furthermore, we wanted to
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class=SpellE>and</span> illustrates that only 6 out of 30 colonies we chose had
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verify that the His Tag we <span class=GramE>added</span> and the Linker
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the insert. </span></p>
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sequence would be exposed enough for us to perform an efficient purification
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and an adequate immobilization. We tested this through the prediction of a
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protein model made using the Software <span class=SpellE>Robetta</span> [1].
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After we took the best model <span class=SpellE>Robetta</span> could offer, we
+
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performed a Structural Alignment using the Human Estrogen Receptor Alpha 2IOG from
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PDB [2] as reference. Our results confirmed that the folding of the protein wasn’t
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being affected at all. <o:p></o:p></span></p>
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<p class=MsoNormal align=center style='text-align:center'><span
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normal'><span style='font-size:15.0pt;line-height:107%;font-family:"Arial",sans-serif;
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<img width=344 height=334
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color:#1155CC;mso-no-proof:yes'><img width=624 height=351
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src="https://2021.igem.org/wiki/images/2/27/T--TecCEM--PartsRegister2image002.jpg"
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alt="Imagen que contiene luz&#10;&#10;Descripción generada automáticamente"
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<p class=MsoNormal align=center style='text-align:center'><b><span lang=EN-US
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1.</span></b><span lang=EN-US style='font-size:10.0pt;line-height:107%;
 
1.</span></b><span lang=EN-US style='font-size:10.0pt;line-height:107%;
 
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color:black;mso-ansi-language:EN-US'> Protein designed by <span class=SpellE>iGEM</span>
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color:black;mso-ansi-language:EN-US'> Gel electrophoresis of the colony PCR we
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<span class=SpellE>TecCEM</span>. It includes the whole receptor <span
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performed for the transformed bacteria BL21 from 1 to 19. We selected only
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class=SpellE>hER</span></span><span lang=EN-US style='font-size:10.0pt;
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those colonies (marked in blue, M8, M13 and M18) that showed the insert at the
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size we expected (1948 bp).</span></p>
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mso-bidi-font-family:"Cambria Math";color:black;mso-ansi-language:EN-US'>&#9082;</span><span
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lang=EN-US style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
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mso-fareast-font-family:"Times New Roman";color:black;mso-ansi-language:EN-US'>
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(red), a linker sequence (green), a His Tag (magenta) and a Signal Peptide
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Sequence (blue). <span class=GramE>It can be seen that both</span> the linker
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sequence and the His Tag are exposed. Protein visualized with Chimera [3].<o:p></o:p></span></p>
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<p class=MsoNormal align=center style='text-align:center'><span
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<p class=MsoNormal align=center style='text-align:center'><b style='mso-bidi-font-weight:
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style='font-family:"Arial",sans-serif;color:black;mso-no-proof:yes'><img width=396 height=348
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normal'><span style='font-size:15.0pt;line-height:107%;font-family:"Arial",sans-serif;
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src="https://2021.igem.org/wiki/images/0/0c/T--TecCEM--PartsRegister3image004.jpg"
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color:#1155CC;mso-no-proof:yes'><img width=624 height=351
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alt="Imagen que contiene collar, luz&#10;&#10;Descripción generada automáticamente"
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src="https://2021.igem.org/wiki/images/a/aa/T--TecCEM--PartsRegister2image004.jpg"
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"Times New Roman";color:black;mso-ansi-language:EN-US'><o:p></o:p></span></p>
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style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
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mso-fareast-font-family:"Times New Roman";color:black;mso-ansi-language:EN-US'></span></p>
  
 
<p class=MsoNormal align=center style='text-align:center'><b><span lang=EN-US
 
<p class=MsoNormal align=center style='text-align:center'><b><span lang=EN-US
 
style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
 
style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
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color:black;mso-ansi-language:EN-US'>Figure 2</span></span><span
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mso-fareast-font-family:"Times New Roman";color:black;mso-ansi-language:EN-US'>Figure
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style='white-space:pre-wrap'></b><span lang=EN-US style='font-size:10.0pt;
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2.</span></b><span lang=EN-US style='font-size:10.0pt;line-height:107%;
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font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";
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EN-US'>. Structural alignment between the protein designed by <span
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color:black;mso-ansi-language:EN-US'> Gel electrophoresis of the colony PCR we
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class=SpellE>iGEM</span> <span class=SpellE>TecCEM</span> (red, magenta, blue
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performed for the transformed bacteria BL21 from 20 to 30. We selected only
−
and green) and the crystallized structure of the Human Estrogen Receptor Alpha
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those colonies (marked in blue, M21, M23 and M26) that showed the insert at the
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2IOG (white). The alignment shows a preserved structure in the Ligand Binding
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size we expected (1948 bp).</span></p>
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Domain of the modified ESR1. </span><span class=SpellE><span style='font-size:
+
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10.0pt;line-height:107%;font-family:"Arial",sans-serif;color:black'>Protein</span></span><span
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style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
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color:black'> <span class=SpellE>visualized</span> <span class=SpellE>with</span>
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−
<span class=SpellE>Chimera</span> [3].</span><o:p></o:p></span></p>
+
  
 
<p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;line-height:107%;
 
<p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;line-height:107%;
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font-family:"Arial",sans-serif;color:black;mso-ansi-language:EN-US'>We also
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font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";
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performed a Molecular Docking to verify its affinity towards a group of common
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color:black;mso-ansi-language:EN-US'></span></p>
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EDCs: Estradiol (the natural ligand), Benzene, Bisphenol A, Dimethyl Phthalate,
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−
Ethylene Glycol, Phenol, Chloroprene and Perfluorooctanoic Acid. To prepare the
+
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model for the analysis, we added hydrogens to it, improving the quality of the
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assay. To this end, we used the tool <span class=SpellE>MolProbity</span> [4].
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The ligands were obtained from PubChem [5] and the Docking was conducted using
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the Software tool <span class=SpellE>SwissDock</span> [6]. From the best result
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of the docking, we obtained its Free Gibbs Energy and used it to calculate the
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Association Constant. We also analyzed the type of interaction. Furthermore, we
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evaluated the formation of hydrogen bonds between the receptor and the small
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molecule. The results are seen below.<o:p></o:p></span></p>
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<p class=MsoNormal align=center style='text-align:center'><b><span lang=EN-US
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<p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;line-height:107%;
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style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
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font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";
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color:black;mso-ansi-language:EN-US'>Table I.</span></b><span lang=EN-US
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color:black;mso-ansi-language:EN-US'>After that, we propagated the selected
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style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
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colonies on liquid LB+CAM medium and took samples of all the cultures at 1.5,
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color:black;mso-ansi-language:EN-US'> Results obtained from the Docking
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3, 4.5 and 48 hours after their inoculation. We ran an SDS-PAGE electrophoresis
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Analysis. Estradiol (highlighted) is the natural ligand of the receptor.<o:p></o:p></span></p>
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expecting to see a protein of around 69.5 <span class=SpellE>kDa</span>
 +
(corresponding to the size of the protein receptor ESR1) but discovered that
 +
only the colonies M18 and M23 had produced a visible protein at the expected site.
 +
The best result corresponded to M23, which we describe below. </span></p>
  
 
<p class=MsoNormal align=center style='text-align:center'><span
 
<p class=MsoNormal align=center style='text-align:center'><span
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style='mso-no-proof:yes'><img width=576 height=448
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style='font-family:"Arial",sans-serif;color:black;mso-no-proof:yes'><img width=545 height=268
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src="https://2021.igem.org/wiki/images/9/91/T--TecCEM--PartsRegister3image006.jpg"
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src="https://2021.igem.org/wiki/images/f/fa/T--TecCEM--PartsRegister2image006.jpg" v:shapes="Imagen_x0020_5"></span><span
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alt="Tabla&#10;&#10;Descripción generada automáticamente" v:shapes="Imagen_x0020_6"><![endif]></span><span
+
 
lang=EN-US style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
 
lang=EN-US style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
−
mso-fareast-font-family:"Times New Roman";color:black;mso-ansi-language:EN-US'><o:p></o:p></span></p>
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mso-fareast-font-family:"Times New Roman";color:black;mso-ansi-language:EN-US'></span></p>
−
 
+
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<p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;line-height:107%;
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font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";
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color:black;mso-ansi-language:EN-US'>To characterize the DNA fragment of ESR1
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we inserted our gene in pSB1C3 and transformed it in <i>E. coli</i> DH5&#945;.
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−
To verify that the insert was cloned correctly, we extracted the plasmid from a
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DH5&#945; culture and performed a PCR using M13 primers. We got positive results
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shown below. <o:p></o:p></span></p>
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<p class=MsoNormal align=center style='text-align:center'><b style='mso-bidi-font-weight:
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normal'><span style='font-family:"Arial",sans-serif;color:black;mso-no-proof:
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yes'><img width=624 height=351
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src="https://2021.igem.org/wiki/images/f/f8/T--TecCEM--PartsRegister3image008.jpg"
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alt="Imagen que contiene Diagrama&#10;&#10;Descripción generada automáticamente"
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v:shapes="Imagen_x0020_7"><![endif]></span></b><span lang=EN-US
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style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
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mso-fareast-font-family:"Times New Roman";color:black;mso-ansi-language:EN-US'><o:p></o:p></span></p>
+
  
 
<p class=MsoNormal align=center style='text-align:center'><b><span lang=EN-US
 
<p class=MsoNormal align=center style='text-align:center'><b><span lang=EN-US
 
style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
 
style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
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color:black;mso-ansi-language:EN-US'>Figure 3</span></b><span lang=EN-US
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mso-fareast-font-family:"Times New Roman";color:black;mso-ansi-language:EN-US'>Figure
−
style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
+
3. </span></b><span lang=EN-US style='font-size:10.0pt;line-height:107%;
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color:black;mso-ansi-language:EN-US'>. Amplification of the ESR1 fragment to
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font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";
−
verify a correct cloning in a pSB1C3 vector. <o:p></o:p></span></p>
+
color:black;mso-ansi-language:EN-US'>SDS-PAGE electrophoresis showing the apparition
 +
of a protein of around 70 <span class=SpellE>kDa</span> which corresponds to
 +
ESR1.</span></p>
  
−
<p class=MsoNormal align=center style='text-align:center'><span lang=EN-US
+
<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US;mso-no-proof:
−
style='font-size:10.0pt;line-height:107%;font-family:"Arial",sans-serif;
+
yes'></span></p>
−
mso-fareast-font-family:"Times New Roman";color:black;mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
+
  
−
<p class=MsoNormal><span lang=EN-US style='font-size:10.0pt;line-height:107%;
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<p class=MsoNormal><span lang=EN-US style='mso-ansi-language:EN-US'></span></p>
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font-family:"Arial",sans-serif;mso-fareast-font-family:"Times New Roman";
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color:black;mso-ansi-language:EN-US'><o:p>&nbsp;</o:p></span></p>
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</div>
 
</div>

Revision as of 16:24, 21 October 2021


ESR1 with Linker, His Tag and Signal Peptide

This sequence codes for the Human Estrogen Receptor Alpha. It was modified in order to express the ligand binding domain and the DNA binding domain. The sequence also includes a 6X Histidine Tag (for its purification), a linker sequence of 4 glycines, 1 serine and 1 cistein (for its immobilization) and a signal peptide sequence (NPS4). This protein is a natural ocurring receptor found in human cells that acts as an enhacer protein which activates after its binding with its ligand. ESR1's natural ligand is Estradiol and after the molecule binds in its domain, it will undergo structural changes that activate the receptor by the formation of a dimer. It has been established that the most important aminoacids for the correct binding of estradiol and ESR1 are Gly521, His524, Leu525 and Met528.

For the purification of ESR1, we added a 6X His Tag and for the immobilization of the protein in a piezoelectric sensor, we added a linker which includes a sequence of 4 glycines, 1 serine and 1 cistein. The addition of the cistein is very important since the immobilization method we chose is based on the formation of a disulfide bond. Furthermore, we chose to mantain the DNA binding domain because it has a region rich in cisteins. Finally, we added a signal peptide sequence NPS4 (which comes from BBa_K3606042), in order to guarantee the secretion of our protein and make it easier for us to purify.

To verify that ESR1 would fold correctly and there would be no steric obstacle, we simulated the protein with its corresponding additions using the tool Robetta (https://robetta.bakerlab.org/). When we recieved the model, we made an structural alignment using the natural protein as reference with a PDB code 2IOG (Protein Data Bank, rcsb.org). Therefore, we concluded that the addition of the Linker, His Tag and Signal Peptide were not interfering with the correct folding of ESR1. Furthermore, we proved that the His Tag and the linker were exposed, so we could use them for the purposed we established previously.

Characterization of BBa_K3809012

For the characterization of the protein BBa_K380912 we transformed the plasmid in E. coli BL21. We then examined the growth of different colonies and performed a Colony PCR to determine if they had the insert or not. The result can be seen in Figure 1. And 2. and illustrates that only 6 out of 30 colonies we chose had the insert.

<img width=624 height=351 src="T--TecCEM--PartsRegister2image002.jpg" alt="Imagen que contiene Tabla Descripción generada automáticamente" v:shapes="Imagen_x0020_3">

Figure 1. Gel electrophoresis of the colony PCR we performed for the transformed bacteria BL21 from 1 to 19. We selected only those colonies (marked in blue, M8, M13 and M18) that showed the insert at the size we expected (1948 bp).

<img width=624 height=351 src="T--TecCEM--PartsRegister2image004.jpg" alt="Imagen que contiene Tabla Descripción generada automáticamente" v:shapes="Imagen_x0020_4">

Figure 2. Gel electrophoresis of the colony PCR we performed for the transformed bacteria BL21 from 20 to 30. We selected only those colonies (marked in blue, M21, M23 and M26) that showed the insert at the size we expected (1948 bp).

After that, we propagated the selected colonies on liquid LB+CAM medium and took samples of all the cultures at 1.5, 3, 4.5 and 48 hours after their inoculation. We ran an SDS-PAGE electrophoresis expecting to see a protein of around 69.5 kDa (corresponding to the size of the protein receptor ESR1) but discovered that only the colonies M18 and M23 had produced a visible protein at the expected site. The best result corresponded to M23, which we describe below.

<img width=545 height=268 src="T--TecCEM--PartsRegister2image006.jpg" v:shapes="Imagen_x0020_5">

Figure 3. SDS-PAGE electrophoresis showing the apparition of a protein of around 70 kDa which corresponds to ESR1.


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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 48
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1264