Difference between revisions of "Part:BBa K4260001"

Latest revision as of 16:02, 12 October 2022

ESR1: Estrogen Receptor 1 with periplasmic signal peptide OmpA, GGGGSC linker and histidine tag

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]

ESR1_HD shortlist resume
Function	Periplasmic expression of Human Estrogen Receptor Alpha protein
Optimization for	E.coli strains
Signal Peptide	OmpA-periplasmic expression
Linker	GGGGSC - create disulfide bonds with chitosan
Added tags	Histidine tag for Nickel Column
Submitted by	Hydro-Defense TecCEM 2022 [1]

Design

The TecCEM team 2022 designed this sequence for the codification of the Human Estrogen Receptor Alpha (hERa). A receptor protein which aim is to bind estrogens and that has affinity to Endocrine Disrupting Chemicals due to important amino acids [2]. Therefore, we used the genomic coding sequence of Homo sapiens Estrogen Receptor 1 (ESR1) optimizing its codons and added features for expression. Thus, a linker composed of four glycines, one serine and one cysteine [3][4] was added as well with the purpose of attaching hER alpha to chitosan and ensure a desired position of the molecule showing the estrogen binding sites up for an effective capture. The signal peptide element helps the cellular machinery to speed up the process of protein expression and send it to the periplasmic space [5][6] , where it can be purified using the histidine tag for a nickel affinity column [6]. Table 1 and figure 1 gives the detailed design of this part.

Figure 1. Construct sequence design.

Sources, usage and biology

Fig.2:Protein ESR1 complexed to estradiol. PDB 1a52 for visualization only. Taken from 10.2210/pdb1A52/pdb

Coded protein

Name: Estrogen Receptor Alpha

Origin: Homo sapiens

Synonyms:ER; ESR; Era; ESRA; ESTRR; NR3A1

Base Pairs: 2111 bp

CDS:coding sequence from nucleotide 232 to 2019 of mRNA from NM_000125.4 isoform 1. [2]

Gene type: protein coding (P03372-UniProt)

Properties:It's affinity to estrogens, estradiol, and endocrine disrupting chemicals.

Nuclear transcription factor whose biological duty is to regulate cellular signaling to enhance physiological processes in humans, in the body it needs hER beta to create a functional complex. For the matter of the project, only the hER alpha is going to be described. ESR1 comes from genomical Homo sapiens ESR1. It contains the elements for coding a protein including its N-terminal ligand transactivation domain, DNA binding domain, hinge domain and the C- terminal ligand transactivation domain (retrieved from NCBI)[8]. hER alphas role is to keep on going the regulation of transcriptional genes inducible by estrogens, thus, enhancing cellular signaling corresponding to metabolic, endocrine, nervous, reproductive systems between others.

Linker

Base Pairs: 18 bp

Linkers are short amino acid sequences that act as spacers between protein domains within a protein. The ones containing Glycines are flexible, separating domains and mostly, creating covalent bonds between proteins. Adding Serine as a polar residue reduces linker protein interaction preserving protein function [3]. Finally, the last residue being cysteine was added to create a disulfide bond with chitosan for surface immobilization, thus keeping the strategy developed by TecCEM 2021 [4][8]

Fig.3:Periplasmic cell space. For visualization only. Created in BioRender

Omp A

Base Pairs: 63 bp

Last but not least, OmpA (Outer membrane protein) signal peptide was retrieved from literature because of its efficiency as periplasmic expression signal peptide [5][6].

Histidine tag

Base Pairs: 18 bp

Histidine tag was chosen for an easy and standardized purification using a Nickel Affinity Column chromatography.[7]

Characterization: protein modeling and molecular docking

Objective

Observe molecular interactions between Human Estrogen Receptor Alpha hER alpha_HD22 coded by BBa_K4260001 and some of its ligands reported in literature such as Estradiol (natural ligand), Carbamazepine, Bisphenol A and Diethyl Phthalate, chemical molecules that acts as Endocrine Disruptors [9]

Methodology

We first modeled our protein sequence hER alpha_HD22 through I-TASSER and the given results were modeled at Chimera, the same as the ligands downloaded from PubChem. We executed the docking hER alpha-ligands using AutoDock Vina and each result was submitted to Protein Plus to observe the interactions between ligands and the protein. Then, returning to the docking, we located these given amino acids to verify if the union matched. The results are shown below.

Protein Model of our designed receptor molecule: Human Estrogen Receptor Alpha (hERα_HD22)

Fig. 4 Molecular simulation of our BBa_K4260001 including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. modeled at Chimera.

Molecular docking between hER alpha and BisphenolA:



Fig 5. BPA molecule, PubChem (6623) and visualized at Chimera.	Fig 6. Interactions between hER alpha and ligand BPA in residues Gly406, Lys407, Phe410, Leu416, Asp417; interactions given by ProteinPlus - Pose view and modeled at Chimera.	Fig 7. Molecular docking of our BBa_K4260001 with one of its ligands BPA (orange) and a G delta of -6.4; including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.

Molecular docking between hER alpha and Carbamazepine:



Fig 8. Carbamazepine molecule, PubChem (2554) and visualized at Chimera.	Fig 9. Interactions between hER alpha and ligand carbamazepine in residues Gly406, Phe410, Leu414; interactions given by ProteinPlus - Pose view and modeled at Chimera.	Fig 10. Molecular docking of our BBa_K4260001 with one of its ligands CBZ (Orange) and a G delta of -8.0; including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.

Molecular docking between hER alpha and Estradiol:



Fig 11. Estradiol molecule, the natural ligand for hER alpha, PubChem (5757) and visualized at Chimera.	Fig 12. Interactions between hER alpha and ligand Estradiol in residues Trp399,Ser474, Leu472,Lys478; interactions given by ProteinPlus - Pose view and modeled at Chimera.	Fig 13. Molecular docking of our BBa_K4260001 with one of its ligands Estradiol (blue) and a G delta of -7.7; including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.

Molecular docking between hER alpha and Diethyl phthalate:



Fig 14. Diethyl phthalate molecule, PubChem (6781) and visualized at Chimera.	Fig 15. Interactions between hER alpha and ligand Diethyl phthalate in residues Tyr136; interactions given by ProteinPlus - Pose view and modeled at Chimera.	Fig 16. Molecular docking of our BBa_K4260001 with one of its ligands diethyl phthalate (blue) and a G delta of -5.8; including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.

Molecular docking of our designed receptor molecule (hERα_HD22) with all the ligands before mentioned:

Fig. 17 Molecular docking of our BBa_K4260001 with its docked ligands; Bisphenol A, carbamazepine, estradiol and diethyl phthalate (moving); including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.
Conclusion

Our protein keeps essential amino acids and regions where ligands such as Bisphenol A, Carbamazepine, Diethyl phthalate and Estradiol have a great affinity. The interactions of these ligands with the amino acids indicated by Protein Plus are observed and confirmed by docking.

Also, in this modeling we observed that a wide range of the ligands' possible interaction residues are not close to the linker and most of them are in the opposite site, leaving that space for the immobilization on chitosan and accommodating the protein as we expect.

References

[1] TecCEM 2022 https://2022.igem.wiki/teccem/ [2] Paterni, I., Granchi, C., Katzenellenbogen, J. A., & Minutolo, F. (2014). Estrogen receptors alpha (ERα) and beta (ERβ): subtype-selective ligands and clinical potential. Steroids, 90, 13–29. https://doi.org/10.1016/j.steroids.2014.06.012

[3] Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi, Jennifer R. Keeffe, Pamela J. Bjorkman. (2014) Design and characterization of structured protein linkers with differing flexibilities. Protein Engineering, Design and Selection, Volume 27, Issue 10, Pages 325–330. https://doi.org/10.1093/protein/gzu043

[4] Chen, X., Zaro, J. L., & Shen, W.-C. (2013). Fusion protein linkers: Property, design and functionality. Advanced Drug Delivery Reviews, 65(10), 1357–1369. doi:10.1016/j.addr.2012.09.039

[5] Goulas T, Cuppari A, Garcia-Castellanos R, Snipas S, Glockshuber R, Arolas JL, et al. (2014) The pCri System: A Vector Collection for Recombinant Protein Expression and Purification. PLoS ONE 9(11): e112643.https://doi.org/10.1371/journal.pone.0112643

[6] https://parts.igem.org/Part:BBa_K208003

[7] IBA Solutions for life sciences. (2021, August). Expression and purification of proteins using 6xHistidine-tag. IBA. https://www.iba-lifesciences.com/media/a8/ee/aa/1631860506/Manual-6xHistidine-tag.pdf

[8] Paterni, I., Granchi, C., Katzenellenbogen, J. A., & Minutolo, F. (2014). Estrogen receptors alpha (ERα) and beta (ERβ): subtype-selective ligands and clinical potential. Steroids, 90, 13–29. https://doi.org/10.1016/j.steroids.2014.06.012 [8] iGEM TecCEM 2021. [9] Ronderos-Lara, J. G. , Saldarriaga-Noreña, H., Reyes-Romero, P. G. , Chávez-Almazán, L. A. , Vergara-Sánchez, J., Murillo-Tovar, M. A. , & Torres-Segundo, C. (2020). Emerging Compounds in Mexico: Challenges for Their Identification and Elimination in Wastewater. In (Ed.), Emerging Contaminants. IntechOpen. https://doi.org/10.5772/intechopen.93909

@@ Line 1: / Line 1: @@
+__NOTOC__
+<partinfo>BBa_K4260001 short</partinfo>
+<partinfo>BBa_K4260001 SequenceAndFeatures</partinfo>
+{| style="color:black" cellpadding="3" cellspacing="1" border="1" align="right"
+! colspan="2" style="background:#C195C4;"|ESR1_HD shortlist resume
+|-
+|'''Function'''
+|Periplasmic expression of Human
+Estrogen Receptor Alpha protein
+|-
+|'''Optimization for'''
+|''E.coli'' strains
+|-
+|'''Signal Peptide '''
+|OmpA-periplasmic expression
+|-
+|'''Linker'''
+|GGGGSC - create disulfide bonds
+with chitosan
+|-
+|'''Added tags'''
+|Histidine tag for Nickel Column
+|-
+|'''Submitted by'''
+|Hydro-Defense TecCEM 2022 [https://2022.igem.wiki/teccem/]
+|}
+===Design===
+The TecCEM team 2022 designed this sequence for the codification of the Human Estrogen Receptor Alpha (hERa). A receptor protein which aim is to bind estrogens and that has affinity to Endocrine Disrupting Chemicals due to important amino acids [2]. Therefore, we used the genomic coding sequence of ''Homo sapiens'' Estrogen Receptor 1 (ESR1) optimizing its codons and added features for expression. Thus, a linker composed of four glycines, one serine and one cysteine [3][4] was added as well with the purpose of attaching hER alpha to chitosan and ensure a desired position of the molecule showing the estrogen binding sites up for an effective capture. The signal peptide element helps the cellular machinery to speed up the process of protein expression and send it to the periplasmic space [5][6] , where it can be purified using the histidine tag for a nickel affinity column [6]. Table 1 and figure 1 gives the detailed design of this part.
+[[File:T--TecCEM--registry-ESR1proteincoding-Design1.jpeg|700px|]]
+Figure 1. Construct sequence design.
+===Sources, usage and biology===
+[[File:T--TecCEM--Registry design Protein ESR1 PDB 1a52.png|250px|thumb|<i><b>Fig.2:</b>Protein ESR1 complexed to estradiol. PDB 1a52 for visualization only. Taken from 10.2210/pdb1A52/pdb</i>]]
+===''Coded protein''===
+'''Name:''' Estrogen Receptor Alpha
+'''Origin:''' ''Homo sapiens''
+'''Synonyms:'''ER; ESR; Era; ESRA; ESTRR; NR3A1
+'''Base Pairs:''' 2111 bp
+'''CDS:'''coding sequence from nucleotide 232 to 2019 of mRNA from NM_000125.4 isoform 1. [2]
+'''Gene type:''' protein coding (P03372-UniProt)
+Properties:It's affinity to estrogens, estradiol, and endocrine disrupting chemicals.
+Nuclear transcription factor whose biological duty is to regulate cellular signaling to enhance physiological processes in humans, in the body it needs hER beta to create a functional complex. For the matter of the project, only the hER alpha is going to be described. ESR1 comes from genomical ''Homo sapiens'' ESR1. It contains the elements for coding a protein including its N-terminal ligand transactivation domain, DNA binding domain, hinge domain and the C- terminal ligand transactivation domain (retrieved from NCBI)[8]. hER alphas role is to keep on going the regulation of transcriptional genes inducible by estrogens, thus, enhancing cellular signaling corresponding to metabolic, endocrine, nervous, reproductive systems between others.
+===''Linker''===
+'''Base Pairs:''' 18 bp
+Linkers are short amino acid sequences that act as spacers between protein domains within a protein. The ones containing Glycines are flexible, separating domains and mostly, creating covalent bonds between proteins. Adding Serine as a polar residue reduces linker protein interaction preserving protein function [3]. Finally, the last residue being cysteine was added to create a disulfide bond with chitosan for surface immobilization, thus keeping the strategy developed by TecCEM 2021 [4][8]
+[[File:T--TecCEM--Registry Design ESR1 Periplasmic cell space.png|thumb|right|250px|<i><b>Fig.3:</b>Periplasmic cell space. For visualization only. Created in BioRender</i>]]
+===''Omp A''===
+'''Base Pairs:''' 63 bp
+Last but not least, OmpA (Outer membrane protein) signal peptide was retrieved from literature because of its efficiency as periplasmic expression signal peptide [5][6].
+===''Histidine tag''===
+'''Base Pairs:''' 18 bp
+Histidine tag was chosen for an easy and standardized purification using a Nickel Affinity Column chromatography.[7]
+===Characterization: protein modeling and molecular docking===
+<html>
+<br><strong><font size=3>Objective</font></strong></br>
+<p align = "justify">Observe molecular interactions between Human Estrogen Receptor Alpha  hER alpha_HD22 coded by BBa_K4260001 and some of its ligands reported in literature such as Estradiol (natural ligand), Carbamazepine, Bisphenol A and Diethyl Phthalate, chemical molecules that acts as Endocrine Disruptors [9]</p>
+<strong><font size=3></font>Methodology</strong>
+<p align = "justify">We first modeled our protein sequence hER alpha_HD22 through I-TASSER and the given results were modeled at Chimera, the same as the ligands downloaded from PubChem. We executed the docking hER alpha-ligands using AutoDock Vina and each result was submitted to Protein Plus to observe the interactions between ligands and the protein. Then, returning to the docking, we located these given amino acids to verify if the union matched. The results are shown below.</p>
+<p align = "justify"><em>Protein Model of our designed receptor molecule: Human Estrogen Receptor Alpha (hERα_HD22)</em></p>
+<br><center><img style="vertical-align: bottom;)" width=30% src="https://static.igem.org/mediawiki/parts/1/1f/T--TecCEM--File-T--TecCEM--results-model-hera-linker_png.png"></center></br>
+<center><strong>Fig. 4</strong> Molecular simulation of our BBa_K4260001 including ESR1 coding sequence (green) and CSGGGG linker (purple). </center>
+<center>Protein designed by TecCEM 2022. modeled at Chimera.</center>
+<br>
+<center>
+<table class="default">
+<tr><th colspan="3" scope="rowgroup"><font size=3>Molecular docking between hER alpha and BisphenolA:</font></th></tr>
+<tr><td></td></tr>
+<tr><td></td></tr>
+<tr>
+<td><img style="vertical-align: bottom;)" width="310" height="310" src="https://static.igem.org/mediawiki/parts/c/c5/T--TecCEM--Results_docking_BPA_molecule_png.png"></td>
+<td><img style="vertical-align: bottom;)" width="310" height="310"src="https://static.igem.org/mediawiki/parts/8/8e/T--TecCEM--results_docking_BPA_interactionsWESR1.png"></td>
+<td><img style="vertical-align: bottom;)" width="310" height="310" src="https://static.igem.org/mediawiki/parts/9/91/T-TecCEM--esr1_verde_bpa.png"></td>
+</tr>
+<tr>
+<td><center><strong>Fig 5.</strong> BPA molecule, PubChem (6623) and <br>visualized at Chimera.</br></center></td>
+<td><center><strong>Fig 6.</strong> Interactions between hER alpha and ligand <br>BPA in residues Gly406, Lys407, Phe410, Leu416,<br> Asp417; interactions given by ProteinPlus - Pose <br> view and modeled at Chimera.</center></td>
+<td><center><strong>Fig 7.</strong>  Molecular docking of our BBa_K4260001 with one of its ligands BPA (orange) and a G delta of -6.4; including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.
+</br></center></td>
+</tr>
+</table>
+</center>
+</br>
+<br>
+<center>
+<table class="default">
+<tr><th colspan="3" scope="rowgroup"><font size=3>Molecular docking between hER alpha and Carbamazepine:</font></th></tr>
+<tr><td></td></tr>
+<tr><td></td></tr>
+<tr>
+<td><img style="vertical-align: bottom;)" width="300" height="300" src="https://static.igem.org/mediawiki/parts/2/25/T--TecCEM--cbz_molecule.png"></td>
+<td><img style="vertical-align: bottom;)" width="300" height="300"src="https://static.igem.org/mediawiki/parts/9/9a/T--TecCEM--interaction_her_alpha_cbz.jpeg"></td>
+<td><img style="vertical-align: bottom;)" width="300" height="300" src="https://static.igem.org/mediawiki/parts/5/52/T--TecCEM--Results_Dckng_ESR1_CBZ_green.png"></td>
+</tr>
+<tr>
+<td><center><strong>Fig 8.</strong> Carbamazepine molecule, PubChem (2554)<br> and visualized at Chimera.</center></td>
+<td><center><strong>Fig 9.</strong>  Interactions between hER alpha and ligand carbamazepine in residues Gly406, Phe410, Leu414; interactions given by ProteinPlus - Pose view and modeled at Chimera.
+</center></td>
+<td><center><strong>Fig 10.</strong> Molecular docking of our BBa_K4260001 with one of its ligands CBZ (Orange) and a G delta of -8.0; including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.
+</center></td>
+</tr>
+</table>
+</center>
+</br>
+<br>
+<center>
+<table class="default">
+<tr><th colspan="3" scope="rowgroup"><font size=3>Molecular docking between hER alpha and Estradiol:</font></th></tr>
+<tr><td></td></tr>
+<tr><td></td></tr>
+<tr>
+<td><img style="vertical-align: bottom;)" width="310" height="310" src="https://static.igem.org/mediawiki/parts/d/d8/T--TecCEM--ESTRADIOL_PNG.png"></td>
+<td><img style="vertical-align: bottom;)" width="310" height="310"src="https://static.igem.org/mediawiki/parts/f/f1/T--TecCEM--Interacciones_estradiol_ESR1.png"></td>
+<td><img style="vertical-align: bottom;)" width="310" height="310" src="https://static.igem.org/mediawiki/parts/8/88/T--TecCEM--results-model-her-linker-estradiol-.png"></td>
+</tr>
+<tr>
+<td><center><strong>Fig 11.</strong> Estradiol molecule, the natural ligand for <br>hER alpha, PubChem (5757) and visualized <br>at Chimera.</center></td>
+<td><center><strong>Fig 12.</strong> Interactions between hER alpha and <br>ligand Estradiol in residues Trp399,Ser474,<br>Leu472,Lys478; interactions given by <br>ProteinPlus - Pose view and modeled <br>at Chimera.
+</center></td>
+<td><center><strong>Fig 13.</strong> Molecular docking of our BBa_K4260001 with one of its ligands Estradiol (blue) and a G delta of -7.7; including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.
+</center></td>
+</tr>
+</table>
+</center>
+</br>
+<br>
+<center>
+<table class="default">
+<tr><th colspan="3" scope="rowgroup"><font size=3>Molecular docking between hER alpha and Diethyl phthalate:</font></th></tr>
+<tr><td></td></tr>
+<tr><td></td></tr>
+<tr>
+<td><img style="vertical-align: bottom;)" width="310" height="310" src="https://static.igem.org/mediawiki/parts/8/8b/T--TecCEM--diethyl-phthalate.png"></td>
+<td><img style="vertical-align: bottom;)" width="310" height="310"src="https://static.igem.org/mediawiki/parts/e/e9/T--TecCEM--ESR1_DIETHYL_sitios_de_interacción.png"></td>
+<td><img style="vertical-align: bottom;)" width="310" height="310" src="https://static.igem.org/mediawiki/parts/3/30/T--TecCEM--results-dckng-esr1-diethylphtalate.png"></td>
+</tr>
+<tr>
+<td><center><strong>Fig 14.</strong> Diethyl phthalate molecule, PubChem (6781) and visualized at Chimera. </center></td>
+<td><center><strong>Fig 15.</strong> Interactions between hER alpha and <br>ligand Diethyl phthalate in residues Tyr136; interactions given by ProteinPlus - Pose view <br>and modeled at Chimera.</center></td>
+<td><center><strong>Fig 16.</strong> Molecular docking of our BBa_K4260001 with one of its ligands diethyl phthalate (blue) and a G delta of -5.8; including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.
+</center></td>
+</tr>
+</table>
+</center>
+</br>
+<center>
+<br><strong><font size=3>Molecular docking of our designed receptor molecule (hERα_HD22) with all the ligands before mentioned:</font></strong></br>
+</center>
+<br><center><video src="https://static.igem.org/mediawiki/parts/b/bd/T--TecCEM--Video_esr1attaching4edcs.mp4" width="640" height="480" controls></video></center></br>
+<center><strong>Fig. 17 </strong>Molecular docking of our BBa_K4260001 with its docked ligands; Bisphenol A, carbamazepine, estradiol and diethyl phthalate (moving); including ESR1 coding sequence (green) and CSGGGG linker (purple). Protein designed by TecCEM 2022. Modeled by AutoDock Vina at Chimera.
+</center></strong>
+<br><strong><font size=3>Conclusion</font></strong></br>
+<p align = "justify">Our protein keeps essential amino acids and regions where ligands such as Bisphenol A, Carbamazepine, Diethyl phthalate and Estradiol have a great affinity. The interactions of these ligands with the amino acids indicated by Protein Plus are observed and confirmed by docking.</p>
+<p align = "justify">Also, in this modeling we observed that a wide range of the ligands' possible interaction residues are not close to the linker and most of them are in the opposite site, leaving that space for the immobilization on chitosan and accommodating the protein as we expect.
+</p>
+</html>
+===References===
+<small>
+[1] TecCEM 2022 https://2022.igem.wiki/teccem/
+[2] Paterni, I., Granchi, C., Katzenellenbogen, J. A., & Minutolo, F. (2014). Estrogen receptors alpha (ERα) and beta (ERβ): subtype-selective ligands and clinical potential. Steroids, 90, 13–29. https://doi.org/10.1016/j.steroids.2014.06.012
+[3] Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi, Jennifer R. Keeffe, Pamela J. Bjorkman. (2014) ''Design and characterization of structured protein linkers with differing flexibilities.'' Protein Engineering, Design and Selection, Volume 27, Issue 10, Pages 325–330. https://doi.org/10.1093/protein/gzu043
+[4] Chen, X., Zaro, J. L., & Shen, W.-C. (2013). ''Fusion protein linkers: Property, design and functionality.'' Advanced Drug Delivery Reviews, 65(10), 1357–1369. doi:10.1016/j.addr.2012.09.039
+[5] Goulas T, Cuppari A, Garcia-Castellanos R, Snipas S, Glockshuber R, Arolas JL, et al. (2014) The pCri System: A Vector Collection for Recombinant Protein Expression and Purification. PLoS ONE 9(11): e112643.https://doi.org/10.1371/journal.pone.0112643
+[6] https://parts.igem.org/Part:BBa_K208003
+[7] IBA Solutions for life sciences. (2021, August). ''Expression and purification of proteins using 6xHistidine-tag''. IBA. https://www.iba-lifesciences.com/media/a8/ee/aa/1631860506/Manual-6xHistidine-tag.pdf
+[8] Paterni, I., Granchi, C., Katzenellenbogen, J. A., & Minutolo, F. (2014). Estrogen receptors alpha (ERα) and beta (ERβ): subtype-selective ligands and clinical potential. Steroids, 90, 13–29. https://doi.org/10.1016/j.steroids.2014.06.012
+[8] iGEM TecCEM 2021.
+[9] Ronderos-Lara, J. G. , Saldarriaga-Noreña, H., Reyes-Romero, P. G. , Chávez-Almazán, L. A. , Vergara-Sánchez, J., Murillo-Tovar, M. A. , & Torres-Segundo, C. (2020). Emerging Compounds in Mexico: Challenges for Their Identification and Elimination in Wastewater. In  (Ed.), Emerging Contaminants. IntechOpen. https://doi.org/10.5772/intechopen.93909
+<small>