Part:BBa_K5107010

hAR/pRR

The human Androgen Receptor coding region utilized for our in-cell biosensor.

Usage and Biology

While the final aim of the project was to develop a cell-free biosensor, multiple stakeholders recommended that we start by trying to create an in-cell system.This was done to test some important aspects of the system. Additionally, another aim of this engineering cycle was to create a system for multiple different receptors. This is necessary to broaden the range of detectable compounds. One of the plasmids that we used to perform our in cell experiments contained the hAR.

Design Phase

The system is used in Saccharomyces cerevisiae (yeast) strain CENPK113-3C, as the receptors have previously been produced successfully in yeast [1]. This is a TRP deficient strain to allow selection for successful transformants. In eukaryotic cells, these nuclear receptors are anchored to chaperones in the cytoplasm. Upon ligand binding, they dimerize, dissociate from chaperones, and translocate to the nucleus where they initiate transcription (facilitated by co-activators) 2, 3. This brought us to the second engineering challenge, concerning the interaction between the receptor and DNA. Literature searches showed us that the receptor response elements do function as singular inverted repeats, but that tandem repeats have been proved to have a much higher effect [1]. Previously, in-cell systems have successfully used five tandem repeats of the REs for different human hormonal receptors [1]. Therefore, the final tandem operator site sequences, denoted ERE5 and HRE5. Those are the elements we used for our cell free system, more details BBa_K5107000 and BBa_K5107001

To complete the design, we had to consider the measurable read-out that could be used for the in-cell system. We landed on a well-characterized beta-galactosidase enzyme as a reporter for the system, inspired by the protocol by Edwards, T. M. et al. (2018) [4]. Using ONPG as a substrate, activity of the β-galactosidase enzyme can be assessed by measuring absorbance at 405 nm.

Build phase

Human Receptor Information

Human Receptor	Response Element (Operator Site)	Natural Hormone	Plasmid Name (In-Cell System)
Estrogen Receptor α (ERα)	ERE	17β-Estradiol	pRR-ERalpha-5Z
Estrogen Receptor β (ERβ)	ERE	17β-Estradiol	pRR-ERbeta-5Z
Glucocorticoid Receptor (GR)	HRE	Dexamethasone	pRR-GR-5Z
Androgen Receptor (AR)	HRE	Testosterone	pRR-AR-5Z
Mineralocorticoid Receptor (MR)	HRE	Aldosterone	pRR-MR-5Z
Progesterone Receptor (PR)	HRE	Progesterone	pRR-PR-5Z

Table 1: Overview of receptors along with corresponding response elements and natural hormones. Finally the plasmid used for the in-cell assay is noted.

The plasmids utilized were found as complete plasmids from Addgene, originally deposited by Miller, Charles A. (2009)[1]. The plasmids were later sequenced by an external company using nanopore. This allowed us to save time we could otherwise have spent cloning. The sequence analysis indicate that the hAR protein sequence is almost identical with the canonical sequence in the Uniprot database for the same protein.

Figure 1:Sequence comparison between the hAR proteins.The first is derived by the Uniprot database, the second is after the sequencing with nanopore technology(made by Unveil Bio)

The plasmids are low-copy CEN/ARS plasmids containing a β-lactamase and a TRP1 gene for selection of successful transformants in E. coli and yeast, respectively. Additionally, they carry one of the hormonal receptors listed in Table 1 transcribed from a GAL1,10 or GPD promoter [1]. Finally, they contain a LacZ gene transcribed from a minimal cytochrome C (CYC) promoter. The receptor response element is located upstream of the CYC promoter. The full plasmid map is shown in Figure 1. below:

Figure 2:Plasmid map of the pRR plasmids utilized in the in-cell system. Contains a LacZ gene encoding β-galactosidase downstream a minimal CYC promoter in which a corresponding receptor response element (RE*: blue) is incorporated. Additionally, this plasmid carries a hormonal receptor (HR*: purple). Finally it carries a TRP1 gene (green) catalyzing tryptophan synthesis, an AMP gene (green) encoding β-lactamase, and CelE1 Ori and CEN/ARS for replication in E. coli and yeast, respectively. Not to scale

The S. cerevisiae strain CENPK113-3C was transformed with the plasmids using a heat-shock method (according to Experiments), which disrupts the cell membranes using LiAc. After the addition of the plasmids, the yeast was grown on a YNB medium without tryptophan to select for successful transformation carrying the TRP1 gene.

The design of the plasmid-based in-cell system is shown in Figure 2 below:

Figure 3:Overview of the plasmid-based in-cell system to detect EDCs. A hormonal receptor (HR) binds the receptor response element (RE) and activates the transcription of a β-galactosidase reporter gene (LacZ) upon binding an EDC. A) shows the system when no EDC is present, while B) shows the system under the presence of an EDC. Not to scale.

Test Phase

To assess whether the system worked, a protocol was designed inspired by the protocol by Edwards, T. M. et al. (2018) [3]. As shown in Figure 2, the β-galactosidase enzyme was used as a reporter of EDCs, and ONPG was used as a substrate, which upon cleavage will absorb at 405 nm. For comparisons between samples, the protocol was based on the principle of measuring the OD610 and OD405 to be able to calculate values independent of the cell density. The complete protocol can be found here. For data handling, the OD values were compared with appropriate media controls (without cells) and vehicle controls (without EDC). The final LacZ value was calculated as the relationship between the OD405 and the OD610 as shown below

Figure 4:Here, t is the incubation time of the enzymatic reaction

‌ The initial tests consisted of creating dose response curves using the natural hormones for the receptors (shown in Table 1). The LacZ values were converted to a percentage of the maximum value for easy comparison between the curves. Unfortunately, we were not able to obtain progesterone to test the PR, and when testing the MR we saw unrealistically high standard deviations and no significant response (data not shown). The four functional dose-response curves are shown in Figure 4 below:.

Figure 5:Dose-response curves for the in-cell assay. Percent maximal response is shown for easy comparison. The concentration of the added hormone is shown - not the concentration in the reaction. All measurements were done in triplicates and standard deviations are represented by error bars. The sample positive controls are shown from left to right; ERb, ERa, AR, GR.

Finally, the system was tested on four water samples. We tested two different types of bottled water, tap water, and a lake sample from a local lake. The data is shown in Table 2 below. As a positive control, the natural hormones (diluted in 50% ethanol) were added to the tap water sample to show that we can replicate the data from the dose-response curves. These positive controls are shown in Figure 4 and fit the dose-response curves nicely.

Table 2: Results from Water Testing

	Bottled water #1	Bottled water #2	Tap water	Lake water
ERα	1.3% ± 5.9%	5.3% ± 3.7%	5.3% ± 3.6%	-0.8% ± 3.0%
ERβ	2.4% ± 16.6%	1.4% ± 6.0%	5.3% ± 4.9%	-1.8% ± 1.8%
GR	-13% ± 50.9%	9.4% ± 11.3%	10.5% ± 10.8%	-2.2% ± 31.5%
AR	15.7% ± 53.4%	28.9% ± 24.7%	28.3% ± 24.0%	33.7% ± 53.0%

Learn Phase

These experiments lead us to conclude three important things. First of all, the system functions, providing an intermediate proof of concept of the in-cell system. Importantly, four of the yeast-produced receptors functioned, and the design with tandem response elements (HRE5 and ERE5) functioned as expected. Secondly, as seen in Figure 3, the biosensor sensitivity differs a lot between receptors. This is however not too important, as the sensitivity for the cell-free assay should be different. Finally, while working with β-galactosidase as a reporter is reliable, it is also a slow and tedious process. Therefore, in the cell-free assay, another reporter method will be utilized.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 2383
  Illegal PstI site found at 166
  Illegal PstI site found at 995
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 2383
  Illegal PstI site found at 166
  Illegal PstI site found at 995
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 2383
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 2383
  Illegal PstI site found at 166
  Illegal PstI site found at 995
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 2383
  Illegal PstI site found at 166
  Illegal PstI site found at 995
  
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Miller, C. A., 3rd, Tan, X., Wilson, M., Bhattacharyya, S., & Ludwig, S. (2010). Single plasmids expressing human steroid hormone receptors and a reporter gene for use in yeast signaling assays. Plasmid, 63(2), 73–78. https://doi.org/10.1016/j.plasmid.2009.11.003
2. Sever, R., & Glass, C. K. (2013). Signaling by Nuclear Receptors. Cold Spring Harbor Perspectives in Biology, 5(3), a016709–a016709. https://doi.org/10.1101/cshperspect.a016709
3. Heery, D. M., Kalkhoven, E., Hoare, S., & Parker, M. G. (1997). A signature motif in transcriptional co-activators mediates binding to nuclear receptors. Nature, 387(6634), 733–736. https://doi.org/10.1038/42750‌
4. Edwards, T. M., Morgan, H. E., Balasca, C., Chalasani, N. K., Yam, L., & Roark, A. M. (2018). Detecting Estrogenic Ligands in Personal Care Products using a Yeast Estrogen Screen Optimized for the Undergraduate Teaching Laboratory. Journal of visualized experiments : JoVE, (131), 55754. https://doi.org/10.3791/55754