Construction Process

As mentioned, the purpose of plasmid construction is to insert ESR1 (which encodes the ERα estrogen receptor) into the prey and bait plasmids, pB42AD and pGilda, respectively.
However, upon doing research and data analysis, we concluded that because BPA and estrogen differ fundamentally in structure, BPA does not bind as effectively to the estrogen receptor (ERα) as estrogen does. This indicates that the interaction might not be strong enough to detect the presence of BPA when we will be able to test it directly from the Y2H system.
To address this, we employed site-directed mutagenesis to enhance the binding affinity between BPA and the ligand-binding domain (LBD) of ERα. Consequently, ESR1 could not be directly inserted into the two plasmids; primers were designed to facilitate cloning of the mutated genes into the vectors.
We chose to divide the ESR1 gene into multiple segments for mutagenesis before cloning rather than ordering fully mutated gene sequences because both approaches require gene selection procedures, and the cost of ordering mutated sequences is approximately nine times higher than performing mutagenesis in segments.

Mutation

ESRα protein consists of 595 amino acids.The original amino acid sequence, N, E, H, K, and P, are located between residue 519 and 535.The new mutant amino acid sequence would be Ｙ,Ｇ,N,T, and L.

Choosing Restriction Sites

Previous studies have demonstrated the efficacy of using EcoRI and BamHI as restriction sites for cloning into the pGlida vector, and EcoRI and XhoI as restriction sites for the pB42AD vector. Accordingly, we adopted these same restriction sites in our experimental design to ensure consistency and reliability.We inserted ESR1 into both the pGlida and pB42AD vectors, with the only difference being the restriction sites used.

ESR1

The gene ESR1 was originally from the pGEM-T vector(3000bp). Previous studies have indicated that the ESR1 300+ fragment possesses important functions; therefore, we cloned the ESR1 300+ fragment separately and compared it with the full-length ESR1.

Cloning

In cloning the full-length fragment, we used the technique of SOE-PCR, the gene was divided into three parts for the PCR amplification: a 917 bp 5′ segment (designated as fragment D), a 687 bp middle segment (fragment B) carrying nine mutations (fragment C was designed solely to introduce an EcoRI restriction site, and the ESR1 gene sequence itself remains identical), and a 243 bp 3′ segment (fragment A) carrying six mutations.
After introducing the mutations into fragments A and B, these two were amplified by PCR and assembled to reconstruct the gene fragment. Fragment D was then amplified and subsequently joined to the AB fragment by PCR, followed by enzymatic digestion and ligation into the plant expression vector.
The cloning strategy was as follows:

• Full-length construct:

1. Assembly of fragments A + B
2. Addition of fragment D to form AB + D

• ESR1 300+ fragment construct:

1. Assembly of fragments A + C (this construct contains only the AC fragments and lacks the upstream A′ region)

The cloning procedures for pGlida and pB42AD were almost identical, except for differences in the restriction sites used.

Plasmid Results

We marked the splicing sites on the plasmids and constructed it like the following diagram.

This is where we got to with our plasmid constructions. To confirm the correct ESR1 insert and orientation in the plasmid, DH5α carrying the plasmid was cultured in antibiotic medium, plasmid was extracted by alkaline lysis, diluted DNA was used for PCR, then sent for Sanger sequencing and verified by BLAST to confirm successful plasmid construction.

Y2H Implementation

After the construction of the plasmids, we input them into our yeast with both the regular set and the mutant set and prepare for the detection system trials.
Normally, the bait’s BD is bound to the upstream operator of the DNA. After BPA enters the yeast cell, it induces the ESR1 regions on both prey and bait to dimerize, letting the two fusion proteins form a transcriptional complex. Next, the prey’s AD recruits RNA polymerase and begins transcription of the downstream reporter gene.

as shown in the prediction image