[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
[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>
<small>
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===TecCEM 2022===
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===Design===
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This construct includes the biobrick (<partinfo>BBa_K4260001</partinfo>) that codes for the ESR1 codon optimized sequence, signal peptide OmpA, linker for positioning the protein in a desired way and a histidine tag for an easy purification. Thus, flanking it with regulatory parts coming from other sources like other team biobricks.
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Therefore, we used the genomic coding sequence as stated in the <partinfo>BBa_K4260001</partinfo> including Homo sapiens Estrogen Receptor 1 (ESR1) optimized for an E.coli expression, with the addition of a (GGGGSC) linker with the purpose of attaching hER alpha protein to chitosan, thus, ensuring the desired position of the molecule showing the estrogen binding site up for an effective capture. The periplasmic signal peptide element “OmpA” helps the cellular machinery to speed up the process of protein expression and send it to the periplasmic space, where it can be purified using the histidine tag for a nickel column.
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===''Coded protein''===
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'''Name:''' Estrogen Receptor Alpha
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'''Origin:''' ''Homo sapiens''
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'''Synonyms:'''ER; ESR; Era; ESRA; ESTRR; NR3A1
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'''Base Pairs:''' 2111 bp
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'''CDS:'''coding sequence from nucleotide 232 to 2019 of mRNA from NM_000125.4 isoform 1. [2]
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'''Gene type:''' protein coding
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Properties:It's affinity to estrogens, estradiol, and endocrine disrupting chemicals.
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Is a 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. The TecCEM team 2022 designed this sequence for the codification of the Human Estrogen Receptor Alpha (hERa), this is a receptor protein whose aim is to bind to estrogens. It is also used as the biological receptor of some endocrine disrupting chemicals.
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[[File:T--TecCEM--Registry design Protein ESR1 PDB 1a52.png|200px|]]
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Figure 2. Protein ESR1 complexed to estradiol. PDB 1a52 for visualization only. Taken from 10.2210/pdb1A52/pdb
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===Regulatory components===
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The regulatory components of this part are;
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- <partinfo>BBa_J64997</partinfo>:Promoter T7 regulates the expression of proteins, commonly used in recombinant
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proteins. It's an efficient element to start transcription and elongation faster than other common
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promoters.
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- <partinfo>BBa_K1624002</partinfo>:Lac Operador is a regulatory element for inducible coding sequences. In the
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absence of lactose, lac repressor occupies the lac operators and prevents transcription, but when lactose or IPTG is
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added, the repressor is absent and the RNA polymerase is free to start the transcription.
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- <partinfo>BBa_K3288007</partinfo>: The Ribosome Binding site according to helps to the recruitment of a
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ribosome during the initiation of the sequence translation.
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- <partinfo>BBa_K395601</partinfo>: Terminator T7 for the ''E.coli'' RNA polymerase.
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===Cultivation, Verification and SDS-PAGE===
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1. The synthetized sequence obtain by IDT, ESR1_HD22, was ligated in pJET 1.2 Blunt vector. We chose this vector because its blunt ends facilitate ligation, resistance to Ampicillin and the killer switch existing inside the vector if the cloning was not correct.
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→ Result: plasmid ESR1_TV (5, 085 bp).
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2. E.coli DH5 alpha strain was transformed with plasmid ESR1_TV for a high plasmid production. We sow on petri plates LB agar adding Ampicillin for the selection marker.
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From the transformed plates; 4 candidate colonies were taken corresponding to ESR1_TV. Striated backups of each colony for further plasmid extractions were done.
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3. Plasmid was extracted from contendant colonies: They were observed in agarose electrophoresis gel to verify integrity and desired size. We noted clear bands of approximately 5 kb, which matched the expected size of our plasmid.
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Result→ Plasmids ESR1_TV (1,2,3,4), refer to '''Fig.3'''.
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[[File:Plas-esr1.png|thumb|center|250px|<i><b>Figure.3:</b> Agarose gel electrophoresis 0.8% running 1Kb extended marker NEB (lane 5), and plasmid ESR1 (1), ESR1 (2), ESR1 (3), and ESR1 (4) extraction from E.coli DH5 alpha.
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</i>]]
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4. For verification: The first PCR was performed using specific primers for our construct amplification; a band size of 2111 bp for ESR1_HD was expected.
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Note: all of our constructs have the same primer homologous region flanking the sequence, this to optimize the use of our reactive in all of our PCR amplifications.
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Bands that correspond to the desired length at lane ESR1_HD (4) were observed in '''Fig.4'''.
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[[File:Pcr-esr1.png|thumb|center|250px|<i><b>Figure.4:</b> Amplification of the construct flanked by our primers region using PCR. Verified through agarose gel electrophoresis 0.8% running 1Kb extended marker NEB (lane 1), and plasmid ESR1 (3) and ESR1 (4). A 2113 bp band shown in ESR1 (4) as expected.
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</i>]]
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5. Digestion of the ligated plasmids “Plasmid ESR1_HD(2,4) ” with restriction enzyme NdeI was carried out to make sure the enzyme cut the plasmid at the desired sites, producing expected fragment sizes.
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Expecting 3936 bp y 1149 bp for ESR1., refer to '''Fig.5'''..
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→Results: the digestion products did match with the theoretical sizes, as shown in fig 13.
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[[File:Dig-esr1.png|thumb|center|250px|<i><b>Figure.5:</b> Digestion with restriction NdeI restriction enzyme, verified through agarose gel electrophoresis 0.8% running 1Kb extended marker NEB (lane 1), and plasmid ESR1 (2) and ESR1 (4).
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</i>]]
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6. E.coli BL21 chemocompetent cells were transformed with “Plasmid ESR1_HD(4)”. Afterwards, inoculation in a selective LB culture medium with Ampicillin was performed for the protein expression and purification. For confirmation that the OmpA signal was working as intended, the periplasmic protein was extracted, and then compared to a control consisting of BL21’s total protein. On '''Fig.6''', even though the control was not as clearly visible as ESR1_HD, there is a band that strongly suggests the presence of the protein, as it fits the expected size, it was found in the periplasmic region and it’s not plainly visible on the control. Even then, we can not assume that the protein observed at that lane is hERa, further experiments must be done before determination
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[[File:Sds-esr1.png|thumb|center|250px|<i><b>Figure.6:</b> SDS-PAGE for verifying presence of hERa in the periplasmic fraction.
[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
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[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
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[5] TecCEM 2021 https://2021.igem.org/Team:TecCEM
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[6] 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
Latest revision as of 16:02, 12 October 2022
ESR1: Estrogen Receptor 1 with periplasmic signal peptide OmpA, GGGGSC linker and histidine tag
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.
[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
[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
