Difference between revisions of "Part:BBa K3737001"

Latest revision as of 01:52, 12 October 2022

Estrogen receptor driven by the T7 Promoter

Added IPTG to activate the RNAP to allow for the expression of the T7 promoter.

Comparing Human and Rainbow Trout Estrogen Receptors - Alma 2022

Estrogen receptors (ER) can bind both the target hormone (17beta-Estradiol, or E2) and other compounds known as xenoestrogens. These other compounds can antagonize the receptor and are often pollutants with important implications for human health, primarily due to their endocrine-disrupting effects. The receptors can likewise form the basis of a biosensor for detection of this type of pollution.

The ERs from different species have different affinities for E2 and various pollutants. In “Differential estrogen receptor binding of estrogenic substances: a species comparison” by Matthews et al (PMID: 11162928 https://pubmed.ncbi.nlm.nih.gov/11162928/), the authors studied binding by displacement. The table below summarizes their findings (IC50 values) and extends it by calculating the Ki or Kd for each xenoestrogen using the Cheng-Prusoff equation. For these experiments, 2.5nM of E2 was used.

(for a table of the IC50, Ki, and relative binding numbers, please visit our Wiki)

Structural comparisons show that binding of molecules such as E2 and DDx result in similar conformations for the Estrogen Receptor. For example, a pairwise structural alignment between hER bound to E2 (PDB ID: 3UUD) and hER bound to DDT (PDB: 5KRA) results in a RMSD of 0.54 – a very close match. For context, an RMSD of 2 is considered excellent resolution when determining a crystal structure.

There is no crystal structure for rtER, so we created a homology model using SWISS-Model (https://www.expasy.org/resources/swiss-model). Below is a comparison of this model, which includes the full protein, to just the ligand binding domain of hER in complex with DDT.

In the above diagram, generated using Mol*, the residues are shaded as hydrophobic (green) to charged (red). As you can see, the binding site on each protein share similar characteristics, with strategic charged residues to complex with the chlorine atoms on DDT.

In a study by Petit et al, “The analysis of chimeric human/rainbow trout estrogen receptors reveals amino acid residues outside of P- and D-boxes important for the transactivation function”, the DNA binding and transcription activity of these receptors was tested (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC102667/). The authors found that rtER transcription activity could only reach 26% of the maximum that could be achieved with hER. Based on their calculations of half-life of the free and complexed molecules, and the resulting Kon and Koff rates that are obtained, a relatively large Kd value of 0.46M can be obtained for rtER. DNA binding by hER was stronger and they could not calculate the half-life of the complex.

Characterizing Expression - Alma 2021

After creating this BioBrick and confirming its sequence, we wanted to find out if bacteria could grow and express it. Attempts to create other estrogen receptors, such as that from Humans, has proven to be toxic to bacteria (e.g. see part BBa_K123003)

Since this Biobrick requires the T7 polymerase for expression, we transformed it into Nico21-DE3 strain from New England Biolabs.

This strain was able to grow, and bacteria continued to grow (the OD increased) after addition of 1mM IPTG to the culture - this indicates that the gene is not toxic.

qPCR results show that the RNA is expressed - crude nucleic acid lysates were subjected to treatment with Reverse transcriptase, or with a control. Following this treatment, they were used in a qPCR with primers specific for this gene.

In a preliminary experiment, the Ct value for the treatment with RT was lower than the control - a difference of 8.13 versus 9.25; this indicates that including the RNA in the cell leads to a 2.17-fold higher amount of nucleic acid. Therefore, we conclude that this gene is being transcribed. Subsequent experiments showed that this expression is dependent on induction with IPTG.

Finally, we performed SDS PAGE on whole cell lysates for cultures with or without 1mM IPTG (grow to steady state, overnight). Preliminary results indicate the presence of a band in a range that is expected of our gene. Together, we conclude that this part works as intended, and produces at least a bit of the estrogen receptor while allowing the bacteria to live.

Here, we used the low molecular range protein molecular weight marker (MWM) from Simga. Highlighted are the 66kDa and 55kDa markers, which are similar in size to our expected product (1800nt ~ 600aa ~ 60kDa).

Sequence and Features