Part:BBa_K4260002:Design

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

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
INCOMPATIBLE WITH RFC[12]
Illegal NheI site found at 1997
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

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.

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