Part:BBa_K4260002
ESR1: Estrogen Receptor 1 coding cassette (Composite part)
| ESR1_HD cassette shortlist resume | |
|---|---|
| Compatible with | RFC 10 |
| Function | Periplasmic expression of Human
Estrogen Receptor Alpha protein |
| Constitutive or inducible | Inducible bye IPTG |
| Optimization for | E.coli strains |
| Submitted by | [1] |
The TecCEM team 2022 [1]designed the biobrick construct cassette for the IPTG inducible expression of the Human Estrogen Receptor Alpha (hERa), this is a receptor protein which aim in our biosensor project is to bind to endocrine disrupting chemicals. Therefore, we used the genomic coding sequence of Homo sapiens Estrogen Receptor 1 (ESR1) adding a linker (GGGGSC) to ensure its binding to chitosan and a histidine tag for a nickel column purification. Its expression was set under control of the T7 promoter and lac operator being inducible by IPTG. Finally, we added a periplasmic signal peptide Omp A. The whole sequence expresses the receptor protein with a linker, setting an specific rotation so that the ligand binding domain (LBD) to be in a reachable position. This ensures the correct positioning of the molecule and therefore, an efficient capture of the EDCs.
Figure 1. Construct sequence design.
Usage and Biology
| ESR1_HD shortlist resume | |
|---|---|
| Expression | OmpA - Periplasmic expression of Human
Estrogen Receptor Alpha protein |
| Regulatory elements | Lac operator and RBS |
| Coded protein | Human Estrogen Receptor alpha |
| Added tags | Histidine tag for Nickel Column purification |
| Submitted by | [1] |
Design
This construct includes the biobrick (BBa_K4260001) 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.
Therefore, we used the genomic coding sequence as stated in the BBa_K4260001 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.
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
Properties:It's affinity to estrogens, estradiol, and endocrine disrupting chemicals.
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.
Figure 2. Protein ESR1 complexed to estradiol. PDB 1a52 for visualization only. Taken from 10.2210/pdb1A52/pdb
Regulatory components
The regulatory components of this part are;
- BBa_J64997:Promoter T7 regulates the expression of proteins, commonly used in recombinant proteins. It's an efficient element to start transcription and elongation faster than other common promoters. - BBa_K1624002:Lac Operador is a regulatory element for inducible coding sequences. In the absence of lactose, lac repressor occupies the lac operators and prevents transcription, but when lactose or IPTG is added, the repressor is absent and the RNA polymerase is free to start the transcription. - BBa_K3288007: The Ribosome Binding site according to helps to the recruitment of a ribosome during the initiation of the sequence translation. - BBa_K395601: Terminator T7 for the E.coli RNA polymerase.
Cultivation, Verification and SDS-PAGE
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. → Result: plasmid ESR1_TV (5, 085 bp).
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. From the transformed plates; 4 candidate colonies were taken corresponding to ESR1_TV. Striated backups of each colony for further plasmid extractions were done.
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. Result→ Plasmids ESR1_TV (1,2,3,4), refer to Fig.3.
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. 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. Bands that correspond to the desired length at lane ESR1_HD (4) were observed in Fig.4.
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. Expecting 3936 bp y 1149 bp for ESR1., refer to Fig.5.. →Results: the digestion products did match with the theoretical sizes, as shown in fig 13.
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
References
[1] TecCEM 2022
[2] NCBI Gene ID: 2099 https://www.ncbi.nlm.nih.gov/gene/2099
[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] TecCEM 2021 https://2021.igem.org/Team:TecCEM
[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
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