Proof of concept
What is a suitable „proof of concept” of our sensor?
While planning experiments, the first question we asked ourselves was: “What actually is a suitable proof of concept of our sensor?”. Of course, at some point, comprehensive studies, including animal models, will be needed to really proof the sensor concept is working. But it was clear to us that these experiments are not the ones to start with. Rather, one first has to study the sensor’s functionality in a culture dish. By this, it can be demonstrated if the sensor is likely to work in a relevant context. Consequently, we thought about the different parts our sensor’s genetic construct is composed of and how they should functionally behave. While successful cloning was always verified by sequencing analysis, the right DNA sequence alone does not prove that genes will actually be expressed by mammalian cells as expected. With this in mind, we planned our proof of concept experiments. Since, in our future vision, our sensor will consist of human intestinal epithelial cells (see Proposed Implementation), we decided to conduct our proof of concept using the human epithelial cell line HeLa. For all the experiments, HeLa cells were transfected with a respective genetic construct and subsequent analysis of expression strength was conducted by appropriate methods.
The following list shows the necessary steps to proof that our sensor concept is working:
Expression of reporter proteins
First of all, we validated that human cells express both reporter proteins chosen for our sensor concept, namely MagAcreated part:
BBa_K3338000 and Gaussia Luciferasecreated part:
BBa_K3338001. For these experiments, genetic constructs carrying either reporter gene under the control of the constitutively active CMV promoterused part:
BBa_I712004 were generated and introduced into HeLa cells. To enable the analysis of MagAcreated part:
BBa_K3338000 expression by fluorescence microscopy, the MagAcreated part:
BBa_K3338000 gene was fused to a gene coding for a fluorescent protein (EGFPused part:
BBa_K1123017). The expression of Gaussia Luciferasecreated part:
BBa_K3338001 was analyzed via luminescence assay. With these experiments, we demonstrated the expression of both reporters in HeLa cells. MagAcreated part:
BBa_K3338000 was shown to be localized within the cells, accumulating in membranous regions. Gaussia Luciferasecreated part:
BBa_K3338001, on the other hand, was detected in the cell culture supernatant. For further details, please refer to our sections of Engineering and Results.
Since our sensor concept is supposed to rely on not only one but two reporter systems, the sensor has to be able to simultaneously express both reporter proteins under the control of the same promoter. Different biological principles lead to such an effect, e.g. the integration of an IREScreated part:
BBa_K3338004 or a P2Acreated part:
BBa_K3338003 site as connecting part between both reporter genes in the sensor’s genetic construct. We therefore examined, which option would yield the best result in regard to a stoichiometric expression.
As the detection of MagAcreated part:
BBa_K3338000 and Gaussia Luciferasecreated part:
BBa_K3338001 requires different readout methods, a direct comparison of the proteins’ expression strengths is not possible. Due to this, we used two fluorescent proteins (EGFPused part:
BBa_K1123017 and mCherryused part:
BBa_J04450) so that analysis can be performed by fluorescence microscopy. Comparing the fluorescence signal intensities of both proteins in the case of usage of an IREScreated part:
BBa_K3338004 or P2Acreated part:
BBa_K3338003 site, respectively, it was shown that the integration of a P2Acreated part:
BBa_K3338003 site lead to a more similar expression strength of both proteins. This is why we decided to use a P2Acreated part:
BBa_K3338003 site for our final sensor construct. For further details, please refer to our sections of Engineering and Results.
Functionality of Gaussia Luciferasecreated
part:
BBa_K3338001
Besides showing that the reporter proteins are expressed in HeLa cells, it is also important to
demonstrate they are actually functional
Since we detected Gaussia Luciferasecreated part:
BBa_K3338001 by performing a luminescence assay, the proof of expression represents a proof of functionality as well. This is due to the fact that only a functional Gaussia Luciferasecreated part:
BBa_K3338001 works as an enzyme that can convert its substrate coelenterazine, thereby producing luminescence. As we performed this assay using cell culture supernatant as sample, we also showed that Gaussia Luciferasecreated part:
BBa_K3338001 is secreted by the cells as expected. For further details, please refer to our sections of Engineering and Results.
BBa_K3338000 For proof of expression, we showed that MagAcreated part:
BBa_K3338000 is synthesized by HeLa cells and that it is localized in membranous regions. While this is a good indicator that the protein features transmembrane regions, it is no proof of its expected functionality as iron transporter, thereby enabling magnetization of the cells. Especially to demonstrate this property, we designed a novel microfluidic measuring chamber able to detect magnetic particles. The chamber was validated using iron microparticles and iron-oxide beads but, unfortunately, due to time and Covid-19 laboratory access restrictions, measurement of MagAcreated part:
BBa_K3338000 expressing cells still remains to be done. Moreover, we have planned to examine the magnetized cells via MRI, which also had to be postponed due to Covid-19. For further details, please refer to our sections of Engineering and Results.
Final applicability of our sensing concept
For the characterization of our reporter proteins, we used genetic constructs with the genes under control of the CMV promoterused part:BBa_I712004. Due to its constitutive activity and thus rather high basal expression level as well as its unresponsiveness to inflammatory signals, the CMV promoterused part:
BBa_I712004 does not fit the demands of our sensor concept. Therefore, we tested different promoters regarding these features.
For our sensor concept, it is extremely important that the expression of our reporter proteins is increased in response to bacterial toxins. To make this possible, the promoter has to be responsive to certain transcription factors such as NF-κB or AP-1, which are activated after binding of inflammatory signals to Toll-like receptors on the cell’s surface. The bacterial endotoxin lipopolysaccharide (LPS) represents such an inflammatory signal which can be generated in the course of biofilm formation. We therefore treated cells, which were previously transfected with the sensor’s genetic construct, with different concentrations of LPS and subsequently analyzed the reporter’s expression strength. With these experiments, we showed that the IL6-promotercreated part:
BBa_K3338008’s activity can be significantly increased after LPS stimulation, demonstrating its inducibility by LPS. For further details, please refer to our sections of Engineering and Results.
Since we do not want our reporter proteins to be constitutively expressed in the human body, the promoter controlling the expression should exhibit a low basal activity. We therefore compared the basal expression level of Gaussia Luciferasecreated part:
BBa_K3338001 as reporter protein using genetic constructs with different promoters. Our results demonstrate that the expression level of Gaussia Luciferasecreated part:
BBa_K3338001 is lowest when expressed under control of the IL6-promotercreated part:
BBa_K3338008. For further details, please refer to our sections of Engineering and Results.
Taken together, our results show that our sensor can be applied to detect the bacterial endotoxin LPS, which is present in all biofilms containing E.coli or other gram negative bacteria, in vitro. This demonstrates a first proof that our sensor concept is likely to work in a relevant context.