Difference between revisions of "Team:Greece/Project"

A therapeutic system needs to be characterized by two crucial features; safety and effectiveness. By giving both features extreme attention, we managed to develop a novel modus operandi, where both the delivery system efficiently targets only cancer cells and the therapeutic RNAi-based logic circuit only affects the specific cancer cell types.

When it comes to colorectal cancer, what better delivery system can one use than the bacteria, that already naturally dwell in the colon? E.coli transformed with a low-copy plasmid containing the part BBa_K1850011 adhere only to colorectal cancer cells and not onto healthy cells, according to iGEM Harvard 2015.

But, in order to improve the system Harvard worked on, we recreated it without the need to control the entire fim operon expression or burden the bacteria with an extra 8kb-plasmid. Finally, going one step further again, we tested the delivery system in a co-culture of bacteria and colorectal cancer cells and succeeded in optimizing the conditions needed for the system to work.

In order to test our colorectal cancer-targeting delivery system, we tested two different strains of E.coli; fimE KO (JW4276-1) and fimH KO (JW4283-3), which were obtained from the Keio collection. The fimE KO is overproducing the type-1 pili, thus we expect them to show extensive adhesion to mannose residues on glycoproteins found on human epithelial cells. Type-1 pili adhesion relies on the fimH lectin which binds to mannosylated glycoproteins, therefore a fimH KO strain is expected to be unable to adhere to epithelial cells. In order to elicit bacterial binding to cancer cells, we transformed them with a plasmid containing the rhamnose inducible fimH 49 KO +RPMrel gene (BBa_K1850011). We then tested our bacteria for their ability to bind to yeast S. cerevisiae, which according to van Asbeck et al. shows a high affinity to mannose binding lectins. The results demonstrated that, when mixed together with yeast, the fimE KO strains appeared to form clumps which precipitated to the bottom of the eppendorf tubes, whilst fimH KO and the fimH KO with the BBa_K1850011 construct did not show any clumps.

Picture 1. Results seen after 1 hour of mixing 500 ul of yeast and E.coli, both grown for 24 hours to saturation. Some minor precipitation has formed in all tubes due to bacterial cells sitting down due to lack of turbulence. * indicates induction with 0.05% rhamnose.

Since the pSB1C3 backbone, in which we received the part BBa_K1850011, is a high copy number plasmid, we wanted to avoid over-expression and the possibility of inclusion body formations by transferring the part to a low copy backbone (pSB1T3) obtained from the part BBa_J04450. This way, our transformed strain possessed both Kanamycin (due to the Keio mutation) and Tetracycline (from pSB1T3) resistance. These bacterial strains were grown in LB Medium with both kanamycin and tetracycline antibiotics and the saturation OD was half (OD600 1.5) compared to the one of fimH KO and fimE KO strains (OD600 3). In order to have comparative results, we diluted the culture in half (OD600 1.5) and the results remained the same.

To validate the expression of the BBa_K1850011 construct, we grew three fimH KO cultures; two were transformed with the RPMrel containing construct, one of which was induced with rhamnose and the last one was transformed with an RFP insert in the same vector as a negative control. Samples were collected at specific time points from each culture, sonicated, centrifuged and finally we separated the supernatant that contains the correctly folded proteins from the pellet. We did a Bradford assay to determine the protein concentration of each supernatant. Afterwards, we run on an SDS-Page 25 ug of the total protein from each sample. The image below shows the SDS-Page, stained with Coomasie G250.

According to the literature, we expect the fimH protein to appear at 30 kDa. Because the pRha has been reported to be leaky, a low intensity band is most likely to appear even in the uninduced samples. We also want to prove that increasing the duration of Rhamnose induction will result in more fimH KO – RPMrel production. To do so, we performed an anti-His Western Blot taking advantage of the his-tag located in the C-terminus of our construct. The fimH KO - RFP sample was used as a negative control. The image below shows the results after ECL staining.

Samples taken at 4 timepoints show increasing expression of fimH in the induced culture. As expected, the uninduced culture appears to have a low intensity band at all timepoints due to the leakiness of the promoter. There is no band on the negative control sample.

Our next goal was to examine whether fimH KO – RPMrel precipitates after induction with Rhamnose. The formation of inclusion bodies containing unfolded protein was observed through a second western blot in which we noticed a very intense band at 30 kDa in the pellet as shown in the picture bellow.

For the first time, instead of using indirect means such as dot blotting (iGEM Harvard 2015) or immunocytochemistry between cells and peptides (Kelly et al., 2003), we proved that the cancer-targeting adhesion system works, using direct means of co-culturing E.coli and Caco-2 cells (human epithelial colorectal adenocarcinoma cells). The RPMrel oligopeptide has been shown by Kelly et al. to bind to 5 specific cancer cell types (HT29, CaCo-2, RKO, SW480 and DLD-1), thus we worked with one of them; Caco-2. For this adhesion test we used the same strains as the agglutination assay. We, again, expected fimE to play the role of the positive control as Caco-2 cells do not lack the natural mannosylated glycoproteins found in epithelial cells. Also, we did not expect the fimH KO mutants to attach to the cells at all, whilst the fimH KO strain with the BBa_K1850011 construct in a low copy backbone was expected to bind. The co-culture protocol was a modified version of Tatsuno et al. and after several trials with various MOI, cell confluences and co-culture incubation times, we managed to standardize the procedure, where both fimE and fimH KOs perfectly served their roles as positive and negative controls respectively, whilst the fimH KOs with the fimH 49 KO+RPMrel clearly showed adhesion to the cancer cells.

The fimE KO strains show visible rings around the Caco-2 cells. In the plates were fimH KO bacteria were inserted, there were almost no bacteria found in the well even after the first washing steps of the immunocytochemistry. The fimH KO strains with the fimH 49KO+RPMrel gene, without rhamnose induction, showed little but clear attachment to the cells, but if induced with rhamnose for 3 hours, clumps of bacteria formed around the Caco-2 cells, which despite all the washing steps did not detach off the cells! Several pictures were taken from all around the 6-wells and all showed similar results to the ones shown.

Taking into account the results from the Agglutination assay, where the fimH KO with the BBa_K1850011 construct on a low copy backbone did not agglutinate to mannose (therefore at healthy epithelial cells), while at the same time our co-culture showed clear adhesion to Caco-2 cells. We are now confident that our targeted delivery system is completely functional!

Lipofectamine 3000 (ThermoFischer Scientific) was used in experiments with the Caco-2, HEK-293 and A549 cell lines. Approximately 4x10⁴ Caco-2 cells or 1x10⁵ HEK-293 and A549 cells in 1 ml of high-glucose DMEM complete medium were seeded into each well of a 24-well and incubated for 24 hours. Transfections were performed according to the instructions of the manufacturer, using the ratios described in [6] for the different plasmids. Doxycycline was added to a final concentration of 1 ug/ml. After a 16-hour incubation, media containing the lipid transfection complexes were replaced with fresh DMEM media and incubated for 2 days before being analyzed for fluorescence.

A BD FACSCalibur analyzer was used for the flow cytometry experiments of the transfected cells. The cells were prepared by trypsinizing each well with 0,25% trypsin-EDTA, collecting the cells, centrifuging them, removing trypsin and resuspending the pellet in fresh PBS.

For each sample, we measured the percentage of DsRed expressing cells and their geometric mean relative fluorescence intensity compared to untransfected controls. We used a GFP expressing plasmid as a transfection control and measured the percentage of GFP expressing cells as well.

To account for difference in transfection efficiencies between the three cell lines we performed the following transformation, calculating the Cellular Fluorescence Intensity for each sample:
$$CFI(sample)=Ds\mathrm{Re}dFI\frac{\%Ds\mathrm{Re}d+}{\%GFP+}$$ To account for differential promoter activity and leakage between our cell lines, so as to be able to compare our results and gauge the performance of our circuit across cell lines we performed an additional normalization step by dividing the CFI of each sample with the CFI of a control plasmid in that cell line, namely pCMV-DsRed-sfGFP-SV40, obtaining the Normalized Fluorescence Intensity, the measure we use for our circuit output.

The double-inversion module necessary for our circuit function is actuated through a Tet-On system which translates the repression on rtTA caused by the “high markers” ,i.e. the upregulated miRNAs, to a reduced post-transcriptional repression on the output plasmid caused by the synthetic miRNA FF4, which is coded downstream of pTRE and therefore increased output. So, we begun by testing whether our Tet-On system along with our synthetic miRNA FF4 were functional in the absence of miRNA control on rtTA, therefore expecting higher circuit output in the Doxycycline uninduced cells.

As expected, we observe a 4-fold increase in circuit output in the uninduced cells, allowing us to proceed with our experiments.

We proceeded by closely examining the classification performance of three circuit topologies; A, B and C characterized by extensive overlap, between Caco-2, our cell line of interest, and HEK-293 and A549.

In addition, we investigated the effect of altered ratios between the components of the inversion module and demonstrated that the system functions best on 1:1 ratio compared to the 2:1 used most often in Tet-On systems.

As we can see our RNAi-based logic circuits are capable of actuating our pre-programmed response (fluorescence in this case) in a cell-type specific manner! Another striking result is that our model seems to be remarkably successful at predicting the classification performance of a given circuit!

Disclaimer: Due to the strict timetable, not all the experiments have been performed in biological triplicates.