Team:Humboldt Berlin/Outlook
During our project, we were able to do the first important steps on the characterization of our designed drug delivery system. We were able to characterize the maintenance of the membrane potential as well as cell size distribution. And from this point we were able to develop a model that is able to predict the expected lifespan depending on the size of the minicells. However, as science does not always work as expected, we were not able to functionalize the minicells to prove that the system can be used as a delivery system. Here, we want to present our outlook for how we would continue to work on the project if we have the chance to.
In order to be able to actively attach to cancer cells, minicells must possess a system that allows for specific cell recognition and adhesion. For this, we constructed two different systems which allow the surface display of rBC2LCN (BBa_K3861001), the recombinant N-terminus of a Burkholderia cenocepacia lectin, which has been shown to be able to interact specifically with several cell lines with tumorigenic characteristics. Surface display via FimH fusion.
Recently, it was shown that surface display using the type 1 fimbriae is possible. By fusing the C-terminus of FimH at the N-terminus of various eukaryotic proteins, the recombinant proteins could be detected at the surface of the bacteria and were still functional.1 Now, by fusing the rBC2LCN to the C-terminus of FimH for assembly at the distal end of the type 1 fimbriae, we seek to achieve surface display and possibly cancer cell adhesion. Surface display via AIDA fusion.
In a second approach, the rBC2LCN lectin will be fused to the AIDA-I autotransporter derived from pRAIDA2.2
Characterization of secretion and surface display
In both constructs, we have included a Human influenza hemagglutinin tag (HA-tag) which will allow for detection of the surface-displayed construct by either Western Blot or immunofluorescence microscopy. This would allow us for proving that the construct is being secreted and, additionally, to determine at which position the construct is found within the cell. Characterization of specific cell binding.
We planned to characterize cell adhesion by fluorescence microscopy. For this, we have chosen the breast cancer cell line MCF-7 to determine the functionality of our construct. rBC2LCN has been shown to be able to detect and adhere to this cell line.3 During the assay, cells would be treated with a dye that stains their cytoskeleton, while the minicells would express a reporter gene that allows for their visualization. Furthermore, binding would be assessed as well in a control cell line to which binding of rBC2LCN has been shown not to bind. Anti-cancer agent expression and translocation through the Type 3 secretion system.
Design of part encoding for anti-cancer agent and the required secretion signal
We have designed a part encoding for an anti-cancer peptide (Pep8, BBa_K3861010) fused to a secretion signal, that allows for translocation into the mammalian cell through the T3SS of the SPI-1 injectisome. This signal contains the first 167 amino acids of the N-terminus of the effector protein SptP. This signal has been successfully used for the secretion of recombinant proteins through the S. Typhimurium injectisome.4 Expression of anti-cancer agent.
During our project, we were able to characterize the expression of the Escherichia coli lactate inducible promoter (PlldR, BBa_K1847008) in our chassis organism, Salmonella Typhimurium. We planned to regulate the expression of our anti-cancer agent from this promoter, as we aimed to induce expression upon a high concentration of lactate. It has been shown previously that the lactate concentration in the microenvironment of tumors tends to be tremendously higher than in healthy tissue.5 Thus, expression of our anti-cancer agent and therefore its translocation would mostly take place within or near tumor tissue. Characterization of secretion.
For the characterization of the peptide secretion, we planned to use the Nano-Glo extracellular detection system from Promega. We fused Pep8 to the small peptide of the split Nluc, HiBiT (BBa_K3861012). This system would allow for detection of the secretory peptide, as the HiBiT would interact with the LgBiT, the complementary part of the peptide forming a functional luciferase that can convert its substrate and luminescence. Characterization of translocation into MCF-7 cells.
For the assessment of Pep8-HiBiT translocation into mammalian cells, we would transfect the MCF-7 cell line with a vector, which allows for the expression of the LgBiT within the cell. Upon translocation of Pep8-HiBiT, the split luciferase would be able to interact with its counterpart to build a functional luciferase, allowing the quantification of the translocation capability of the peptide.
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Outlook
Outlook
Minicell functionalization