Induce expression of human estrogen receptor (hER) - which is to bind to any oxybenzone present (analogous to estrogen molecule)
Find a mechanism to induce the expression of GFP in the presence of oxybenzone - we do this via the estrogen receptor protein and estrogen response element (DNA sequence)
The pRSET/EmGFP is a bacterial plasmid with two origins of replication (f1 and pUC) and ampicillin resistance gene, which were the basic components we need in our plasmid to ensure successful propagation and selection of transformed E. coli. Additionally, it was already integrated with the EmGFP (Emerald Green Fluorescence Protein) sequence which we need in order to detect and quantify the presence of oxybenzone in water samples to be tested. The plasmid also have many single restriction sites which allow us to clone in our desired genetic construct or other iGEM biobricks easily. Our synthesized DNA sequence contains one direction that synthesizes the human estrogen receptor and the other direction that synthesizes EmGFP when the expressed estrogen receptor binds to any present oxybenzone inside the cell.
In order for oxybenzone to bind to a human estrogen receptor (hER), we first need to produce an estrogen receptor within the E. Coli. We chose to use a T7 promoter as it allows for continuous expression of a gene in prokaryotes. We also added a ribosome binding site (aggaggacagcu) 6 nucleotides before the start codon. A T7 transcription terminator is added to end transcription at the end of the hER gene.
We found that after estrogen binds to the human estrogen receptor (hER), it forms a dimer. As oxybenzone is a xenoestrogen, we assume that it acts the same way and also forms a dimer upon binding to the receptor. In the estrogen transduction pathway, the dimer acts as a transcription factor, by binding to estrogen response elements (EREs) and inducing expression of genes such as peroxidase, cadherin, and adenine nuclear transcription factor [1]. This led us to include the EREs in our plasmid to make the expression of emGFP* responsive to the level of oxybenzone present.
A study published in Molecular Endocrinology found that 4 EREs are required to optimize expression of a reporter gene upstream from a minimal promoter. The study also found that the EREs should be 83 nucleotides away from the promoter and 38 nucleotides space present between each ERE [2]. We used a Pribnow Box (analogous to eukaryotic TATA box) 10 nucleotides upstream of the GFP and a Transcription Control Element 35 nucleotides upstream of the minimal promoter to optimize transcription of our constructs. These two elements together act as a TATA box would act in eukaryotic expression, initiating a very low level of gene expression [3]. Adding the EREs as transcription factors should make expression of the GFP responsive to the levels of oxybenzone present inside the cell. Additionally, we added a ribosome binding site before the GFP gene also 6 nucleotides before the start codon as it is also placed before the hER gene. This basic genetic construct design would then allow us to calculate the oxybenzone level present in the sample based on the fluorescence intensity measured from the induced level of GFP expression.
*emGFP and GFP are used interchangeably
Gene Circuit showing the theoretical flow of gene expression using our constructs.
The ampicillin resistant gene is used to show that the plasmid has successfully been transformed. Only the plasmids containing the antibiotic resistant gene will function properly. The T7 promoter makes the estrogen receptor, to which the oxybenzone binds. Oxybenzone is known for mimicking estrogen, which allows the estrogen receptor to go through a conformational shape change, forming a dimer, and that activates the estrogen response elements. The estrogen response elements increases expression, which allows the Pribnow box to efficiently transcribe EmGFP into GFP.
[1] Estrogen signaling pathway. Estrogen Signaling Pathway - Creative Diagnostics. (n.d.). Retrieved October 4, 2022, from https://www.creative-diagnostics.com/estrogen-signaling-pathway.htm.
[2] Sathya, G., Li, W., Klinge, C. M., Anolik, J. H., Hilf, R., & Bambara, R. A. (1997, December 1). Effects of multiple estrogen responsive elements, their spacing, and location on estrogen response of reporter genes. OUP Academic. Retrieved October 1, 2022, from https://academic.oup.com/mend/article/11/13/1994/2754076
[3] Pribnow Box. Pribnow Box - an overview | ScienceDirect Topics. (n.d.). Retrieved October 4, 2022, from https://www.sciencedirect.com/topics/agricultural-and-biological-sciences/pribnow-box