Contribution | Alma

Our Contributions

We made a number of contributions to the iGEM community and the Registry of Standard Biological parts. This includes more background information on both Human (Homo Sapien) and Rainbow Trout (Oncohynchus mykiss) Estrogen Receptor. This information can be found here, and on the part page for Rainbow Trout estrogen receptor (rtER) under T7 promoter control: BBa_K3737001, and on the page for Human Estrogen receptor: BBa_K123003

The rainbow trout estrogen receptor (K3737000) was selected for this project because it was found in previous research to closely emulate the responses of that of the human estrogen receptor. The sensitivity of the rainbow trout estrogen receptor (henceforth RTER) is slightly higher than the threshold needed for that of the human estrogen receptor, but has a very high resistance to androgenous activation by other hormones such as testosterone or testosterone agonists. It was for this reason that the rainbow trout was chosen, in addition to the fact that it has been shown that the RTER is similarly capable of transcription in a strictly hormonal activation pathway.

Our kill switch (see engineering success) is based upon a quorum sensing module. In order to better design this part, we researched more about the LuxI gene, and included that information on the relevant part page, BBa_C0062. We hope that this information will assist teams that are planning on using LuxI in their experiments, or looking to modify LuxI activity by mutations.

In addition, we provided additional instructions on how to setup cell-free protein synthesis. We followed work by Fujiwara et al, 2016 - which described in detail how to create a cell extract for protein synthesis. This extract contains RNA polymerase and Ribosomes, as well as all factors for translation. However, it must be supplemented by a solution of nutrients, co-factors, and amino acids. Although the paper details the final concentration of this mixture, it does not provide guidance on how to set it up. We found this to be a time consuming process due to the differing solubility requirements. To save future iGEM teams time, we provide the table below (and on our Proof of Concept page)

Finally, we made contributions through some of our measurements - testing and further demonstrating the leaky expression of BBa_J04450, for example, or showing how that and other BioBrick-containing E. coli grow in the presence of anhydrotetracycline, IPTG, and Genistein. Most importantly, we present a new type of model for the expression of BBa_J04450 that takes into account plasmid copy number. We also tested the idea of using a double inverter to preserve system logic and change expression dynamics, which we also modelled. Since these are project agnostic, we hope that they are generally useful to the iGEM community.

Estrogen Receptors

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), 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.

Agonist	IC₅₀ (M)	K_i (M)	RBA	IC₅₀ (M)	K_i (M)	RBA
	Rainbow Trout (rtER). K_d = 6x10^-10			Human (hER). K_d = 4x10^-10
o,p-DDT	1.80x10^-7	3.48x10^-7	0.1148	2.00x10^-4	2.76x10^-5	0.00001
o,p-DDE	3.20x10^-6	6.19x10^-7	0.0065	2.00x10^-4	2.76x10^-5	0.00001
p,p-DDE	8.00x10^-6	1.55x10^-6	0.0026	2.00x10^-4	2.76x10^-5	0.00001
p,p-DDT	2.00x10^-6	3.87x10^-7	0.0103	2.00x10^-4	2.76x10^-5	0.00001
Estradiol (E2)	3.30x10^-9	6.39x10^-10	6.2626	2.90x10^-10	4.00x10^-11	100.00
Genistein	7.50x10^-7	1.45x10^-7	0.0276	6.30x10^-7	8.69x10^-8	0.0460
Tamoxifen	1.30x10^-8	2.52x10^-9	1.5897	2.80x10^-8	3.86x10^-9	1.0357
BPA	1.60x10^-6	3.10x10^-7	0.0129	3.60x10^-5	4.97x10^-5	0.0008

For hER binding to DDx compounds, we used an IC50 of 200uM, since Matthews et al did not observe the IC50 with 100uM.

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.

In the above image, 3UUD (Brown) and 5KRA (Blue) are superimposed. 251/257 of the residues matched, showing a 93% coverage.

There is no crystal structure for rtER, so we created a homology model using 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 (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 K_d 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. According to sources found by the 2020 Alma iGEM team, the DNA binding for hER is much better - a K_d of 1.0x10^-10M!. Direct comparison of the two is limited, but the paper referenced above used 0.08pmol of Estradiol in their experiments.

Additional Sources and References

Le Dréan Y, Kern L, Pakdel F, Valotaire Y. Rainbow trout estrogen receptor presents an equal specificity but a differential sensitivity for estrogens than human estrogen receptor. Mol Cell Endocrinol. 1995 Mar;109(1):27-35. doi: 10.1016/0303-7207(95)03482-m. PMID: 7789614.
R. Le Guével, F.G. Petit, P. Le Goff, R. Métivier, Y. Valotaire, F. Pakdel, Inhibition of Rainbow Trout (Oncorhynchus mykiss) Estrogen Receptor Activity by Cadmium, Biology of Reproduction, Volume 63, Issue 1, 1 July 2000, Pages 259–266,

Recipe for making CFPS solution

In Fujiwara et al, 2016, a method for cell free protein synthesis is described. We are providing more information here - how to make stock solutions of each of the components needed for the amino acids and energy mixture, to be combined with the LoFT extract and the plasmid or PCR DNA. In the table below, the amino acids are listed only by their 1 letter abbreviation. IPTG is included to induce gene expression if you are using a pLAC promoter or a T7 based system (with T7 RNAP under pLac control). According to the original paper, some of these components are dispensable.

	CURRENT CONCENTRATION (M)	DESIRED CONCENTRATION (M)	uL
NAD+	0.5	0.00033	0.66
ATP	0.5	0.0015	3
CTP	0.05	0.0009	18
GTP	0.05	0.0015	30
UTP	0.05	0.0009	18
E	0.005	0.0005	100
W	0.05	0.0005	10
R	0.5	0.0005	1
C	0.5	0.0005	1
S	0.5	0.0005	1
G	0.5	0.0005	1
K	0.5	0.0005	1
P	0.5	0.0005	1
A	0.5	0.0005	1
HEP	1	0.05	50
PEG	0.2	0.02	100
MAL	1	0.012	12
MgACT	1	0.014	14
SPERM	0.003	0.001	333.333333
FA	0.068	0.000068	1
CAMP	0.146	0.00075	5.1369863
TRNA	0.2	0.0002	1
KE	0.5	0.09	180
Y	0.0025	0.0005	200
V	0.25	0.0005	2
F	0.1	0.0005	5
T	0.25	0.0005	2
M	0.1	0.0005	5
H	0.1	0.0005	5
I	0.1	0.0005	5
L	0.1	0.0005	5
N	0.091	0.0005	5.49450549
Q	0.167	0.0005	2.99401198
IPTG	0.1	0.001	10
COA	0.013	0.00026	20
3PGA	0.1075	0.036	334.883721
D	0.1	0.0005	5

Reference: Fujiwara K, Doi N. Biochemical Preparation of Cell Extract for Cell-Free Protein Synthesis without Physical Disruption. PLoS One. 2016 Apr 29;11(4):e0154614. doi: 10.1371/journal.pone.0154614. PMID: 27128597; PMCID: PMC4851396.