Contribution

"If you have knowledge, let others light their candles with it" - Margaret Fuller

SEAP secretion

Characterization

Colorimetric assay

Caculating Activity

Secreted Alkine Phosphatase SEAP

SEAP is a useful reporter protein to investigate the amount of transcriptional activity of enhancer/promoter elements. SEAP arises from the SEAP gene that encodes for a truncated form of human placental alkaline phosphatase (PLAP). This truncated form lacks a crucial membrane-anchoring domain, resulting in efficient secretion of SEAP by transfected cells.1 The levels of secreted SEAP can be derived from the culture medium and are directly proportional to the changes in intracellular concentrations of SEAP mRNA and SEAP protein.1,2


Because of the natural secretion of SEAP, its use as a genetic reporter provides several advantages: 1) the SEAP assay does not require the preparation of cell lysates, 2) during the SEAP activity measurement, the transfected cells remain unharmed, 3) the gene expression kinetics can be studied easily, as the medium of the same cultures can repeatedly be collected, 4) the culture medium does not contain significant background from endogenous alkaline phosphate activity, 5) usage of multi-well plates allows for easy automation of sample collection and assays. Therefore, SEAP reporter systems are appropriate for high-throughput applications.1,2


In our project, the SEAP reporter system is a valuable tool to quantitively measure the activity of the GEMS receptor of our engineered cells (Figure 1). Activation of the GEMS receptor will lead to activation of the Janus Kinase/Signal Transducer and Activator of Transcription (JAK/STAT) pathway. Subsequently, this pathway induces gene expression of the transcription factor Signal Transducer and Activator of Transcription 3 (STAT3). STAT3 will bind its promotor sequence located in a plasmid in front of the SEAP gene, resulting in activation of SEAP gene expression. Altogether, activation of the Generalized Extracellular Molecule Sensor (GEMS) receptor will eventually lead to the secretion of SEAP protein by our engineered cells.

Figure 1 | Design of the SEAP assay using engineered HEK293T cells. The binding of the ligand RR120 to the RR120 VHH domain results in the activation of the GEMS receptor. Through the JAK/STAT pathway, gene expression and secretion of SEAP will be induced.

Characterization of SEAP

The part registry does not yet contain a complete description of the characteristics and advantages of SEAP (BBa_K1470004) as a genetic reporter. However, SEAP is the most commonly used secreted serum reporter to monitor gene expression in eukaryotic cells3. Moreover, while the SEAP assay has clear advantages, the registry does not yet include measurements on the SEAP reporter system obtained by a colorimetric SEAP assay. Therefore, our iGEM team extended the documentation of BBa_K1470004 with an overview of the advantages of using SEAP as a reporter protein. In addition, we added data of SEAP measurements to the registry, supplemented with both an elaborated description and a MATLAB script to calculate the activity of the secreted SEAP from the absorbance that has been measured with a plate reader during the colorimetric SEAP assay. We believe this will greatly help the iGEM community as well as future iGEM teams.


Colorimetric SEAP assay

Before performing the colorimetric SEAP assay, HEK293T cells were seeded on a 24-well plate with each well containing 0.24·106 cells. Subsequently, the HEK293T cells were transfected with pLS13; a mammalian reporter plasmid for STAT3-induced SEAP expression (Ostat3-PhCMVmin-SEAP-pA)4, pLS15; a vector for mammalian STAT3 expression (PhCMV-STAT3-pA)4, and pLeo619-PSV40; a mammalian expression vector to recombinantly express the EpoRm-IL-6RBm-pA fused to the RR120 camelid heavy chain antibody VHHA52 (GenBank accession no. MG437012)5. After the transfected cells were treated with the ligand azo dye RR120, the cells were incubated for 48 hours at 37°C and 5% CO2. Subsequently, the medium was aspirated from the cells, to perform the SEAP assay.


To perform the colorimetric SEAP assay, the Secreted Alkaline Phosphatase Reporter (SEAPorterTM) Assay Kit (Novus Biologicals, catalog# NBP2-25285)6 was used. In this assay, the colorless alkaline phosphatase substrate p-nitrophenyl phosphate (pNPP) is added to the collected medium containing secreted SEAP. As a consequence, SEAP catalyzes the hydrolysis of pNPP (Figure 2).

Figure 2 | Hydrolysis reaction of pNPP catalyzed by SEAP. Adapted from: Dorobanłu et al.7

This reaction results in the yellow-colored p-nitrophenol (pNP). Following the production of this product, the absorbance was measured every half a minute for one hour at a wavelength of 405 nm by the Tecan Spark plate reader. Hereof, the catalytic activity of SEAP was calculated (Figure 3). The figure demonstrates significant SEAP activity after treating the ligand RR120. Compared to the samples to which no RR120 was added, a 32-fold and 34-fold increase in SEAP activity was obtained after treating the cells with 100 ng/ml and 300 ng/ml RR120, respectively. This indicates that activation of the GEMS receptor was obtained after treating the HEK293T cells with RR120.


Figure 3 | SEAP activity calculated from the absorbance at 405 nm measured with the plate reader. HEK293T cells transfected with plasmids encoding for the RR120 VHH GEMS receptor, the STAT transcription factor, and the SEAP reporter protein, were treated with different concentrations of the ligand RR120. After 48 hours of incubation, in which the cells produced SEAP, the cell culture medium was aspirated and a colorimetric SEAP assay was performed. The absorbance at 405 nm was measured every half a minute for 1 hour. Subsequently, from this measurement, the SEAP activity was calculated. n=3 biological replicates were used

Calculating SEAP activity

The absorbance at 405 nm was measured in absorbance units (AU). To determine the SEAP activity, a calibration curve was made by measuring samples containing a known concentration of pNP (Figure 4). This calibration curve is specifically made for the plate reader that has been used to measure the absorbance. Importantly, using a different plate reader requires a calibration curve made for that specific plate reader to allow accurate calculations. As a result, certain absorbance values have been measured for known concentrations of pNP.


Figure 4 | The calibration curve obtained by measuring the absorbance from a titration of known concentrations of pNP. A straight line was fitted through the measured absorbance values. This line was described by the formula: Absorbance [AU] = a · pNP[mM] + b. This calibration curve is specifically obtained for the plate reader that was used for the measurement of the colorimetric SEAP assay.

For all cell culture medium samples, obtained from the cells treated with different concentrations of ligand RR120, the absorbance was measured every half a minute for one hour. Per sample, the calibration curve was used to convert all measured absorbance values into concentrations of the product pNP present in the medium samples. This resulted in a graph containing data points that equal the concentrations of pNP measured over time. Through these data points, a straight line was fitted. The slope of this line determined the conversion of pNPP to pNP by SEAP per minute. As a known concentration of pNPP was added to the sample, the SEAP activity, expressed in enzyme units per liter (U/L), could be calculated from this slope. One enzyme unit equals the enzyme activity by converting 1 μmol of pNPP by SEAP, per minute.8 The MATLAB script our iGEM team used to calculate the SEAP activity can be found here:

Seap-code.pdf
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