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The calcein AM assay could successfully distinguish between dead and alive cells under the selected experimental conditions. Moreover, the killing can also conveniently assessed in HeLa CCL-2 lc cells just by dead-cell removal and assessment of constitutively expressed citrine in the absence of any permeat dye. <br></p> | The calcein AM assay could successfully distinguish between dead and alive cells under the selected experimental conditions. Moreover, the killing can also conveniently assessed in HeLa CCL-2 lc cells just by dead-cell removal and assessment of constitutively expressed citrine in the absence of any permeat dye. <br></p> | ||
</details> | </details> | ||
+ | |||
+ | <details> | ||
+ | <summary>Assessing the bacterial lysate tolerance of different cell lines </summary> | ||
+ | |||
+ | <p><strong>OBJECTIVE</strong><br> | ||
+ | With this experiment we wanted to assess the tolerance of different cell line upon incubation with different amount of azurin-free bacterial lysate. The information acquired from this expreriment helped us to define the amount of medium that can we substitute by our bacterial lysate without harming the cells neither because of bacterial toxins present in the lysate nor because of nutrient depravation. </p> | ||
+ | |||
+ | <p><strong>PROCEDURE</strong><br> | ||
+ | For different cell-lines, namely, HeLa CCL-2, HeLa CCL-2 lc, HT-29 and HEK - 239 cells were seeded in 4 different 12-well culture plates with initial concentration of 100'000 cells/mL. After 24-hour growth the cells are incubated with increasing amounts of azurin-free lysate in order to assess the effect of the lysate on the cancer cell growth . Simultaneously, cell samples were also incubated with increasing amount of PBS, instead of lysate, in order to check the effect of nutrient depravation on the cell growth. After 24 hours the cells, apart from the HeLa CCL-2 lc, were incubated with calcein AM solution. The viability of the cells was assessed via fluorescnce microscopy (Figure 1). The samples of HeLa CCL-2 lc cell-line were direcly examined for their viability via fluorescence microscopy (HeLa CCL-2 lc) as these cells are producing constitutively citrine. A detailed protocol is available in <a href="https://2017.igem.org/Team:ETH_Zurich/Protocols">Protocols.</a> | ||
+ | </p> | ||
+ | |||
+ | <p><strong>RESULTS</strong><br> | ||
+ | From the microscopy pictures obtained, we could verify the apoptotic effect on both the cell lines after 24-hours incubation with the killing agent (Figure 1). | ||
+ | |||
+ | <figure class="fig-nonfloat" style="width:500px;"> | ||
+ | <img alt="staining killing images" | ||
+ | src="FIX ME"> | ||
+ | <figcaption>Figure 6. Pictures of mammalian cell cultures with different 5-fluouracil concentrations. </figcaption> | ||
+ | </figure> | ||
+ | |||
+ | The same results are verified also by the spectrophotometer measurements both for the HeLa CCL-2 stained with calcein AM and the citrince expressing HeLa CCL-2 lc. The killing curves obtained can be shown in the floowing figure (Figure 2). </p> | ||
+ | |||
+ | <figure class="fig-nonfloat" style="width:500px;"> | ||
+ | <img alt="tecan killing measurements" | ||
+ | src="FIX ME"> | ||
+ | <figcaption>Figure 6. Tecan measurements for killing for HeLa and HeLa lc.. </figcaption> | ||
+ | </figure> | ||
+ | |||
+ | <p><strong>CONCLUSION</strong><br> | ||
+ | The calcein AM assay could successfully distinguish between dead and alive cells under the selected experimental conditions. Moreover, the killing can also conveniently assessed in HeLa CCL-2 lc cells just by dead-cell removal and assessment of constitutively expressed citrine in the absence of any permeat dye. <br></p> | ||
+ | </details> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
+ | |||
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</div> | </div> | ||
Revision as of 06:37, 1 November 2017
![CATE logo](https://static.igem.org/mediawiki/2017/d/d6/T--ETH_Zurich--LogoMinimal.png)
Anti-Cancer Toxin Experiments
Introduction
Once a tumor environment has been recognized and colonized by our bacteria, the production of an anti-cancer toxin, together with an MRI Contrast Agent agent will be triggered. The module that we engineered to achieve this purpose includes an AND-logic synthetic promoter that recognizes the presence of high lactate and high bacterial density in the tumor environment and allows the expression of the anti-cancer toxin, azurin, and the MRI Contrast Agent, bacterioferritin, from the same operon (Figure 1).
![Accumulation of anti-cancer toxin into cells](https://static.igem.org/mediawiki/2017/9/9a/T--ETH_Zurich--Wet_Lab_Experiments_Anti_Cancer_Toxin_animation_1.gif)
Once the Tumor Sensing has been activated, both the Anti-Cancer Toxin azurin and the MRI Contrast Agent bacterioferritin are produced. Azurin will accumulate inside of the bacteria until it is ready for release. Once the doctor verifies the bacterial colonization of the tumor bacteria via MRI, focused ultrasounds will activate Cell Lysis releasing an effective dosage of azurin to the tumor.
For more details about azurin and its role in our system, go to our description of the Anti-Cancer Toxin.
Overview of the Experiments
In our experiments, we replaced the AND-gate promoter in the genetic circuit depicted in Figure 1 with a well-established inducible promoter, Plux, in order to control the expression of the operon. Moreover, we coupled the expression of azurin with a fluorescence output as we exchanged the bacterioferritin gene with a gene encoding for superfolder GFP. In that way we could indirectly assess the functionality of our operon in a controllable manner (Figure 2). This circuit was transformed in E. coli TOP10 which was used for the production of azurin-rich bacterial lysate. Finally, the application of the bacterial lysate on different cancer-cell lines in vitro helped us to emulate the delivery of the anti-cancer payload via cell-lysis against different solid-tumor types.
![](https://static.igem.org/mediawiki/2017/6/63/T--ETH_Zurich--Wet_Lab_Experiments_Anti_Cancer_Toxin_fig_2_experiment.png)
Several preliminary experiments were conducted before the application of the bacterial lysate to different cancer-cell lines. Initially we examined the production of azurin from our engineered bacteria after induction with AHL, indirectly by detecting the fluorescence of the co-expressed sfGFP. An SDS-PAGE analysis confirmed the presence of azurin in the cytosolic fragment of E. coli after AHL induction. Further culture scale-up to 50 mL volumes allowed us the production of azurin-rich bacterial lysate via sonication. A second SDS-PAGE confirmed cell-lysis by sonication as all the overexressed azurin and sfGFP were detected together with the native proteins of E. coli within the lysate. Thanks to our collaborators from Freiburg, we were able to quantify the azurin-content of our bacterial lysate, as they provided us with purified azurin, which we used to create a standard curve of purified azurin concentration on the same SDS-PAGE gel.
With our bacterial lysate at hand we had to define the following parameters regarding the experimental set-up of mamalian cell incubation with our lysate:
- Which cancer cell-lines should we use?
- Which is the optimal number of cells to seed in our experiments?
- Which staining assay should we use to assess the viability of the mammalian cells?
- How much of azurin-free bacterial lysate can our cells tolerate?
After defining the experimental parameters we were able to test the cytotoxic potential of our azurin-rich bacterial lysate against different cancer cell-lines. Simultaneously, our Freiburg collaborators provided us with a killing curve of purified azurin against HEK-239 cells, enabling the comparison between the efficacy of purified azurin and azurin - rich bacterial lysate.
To read more about each of these experiments, click on the buttons below. For a detailed protocol describing each experiment, visit Protocols.
Co-expression of azurin and sfGFP by E. coli TOP10
OBJECTIVE
In this experiment we attempted to co-express azurin and sfGFP in the same operon in a controllable manner, emulating the co-expression of azurin and bacterioferitin in our final genetic circuit. The expression of sfGFP was used as an indirect readout of the operon gene expression. Additionally we tried to express a small azurin-derived peptide, namely p28, which is responsible for azurin's cytotoxic effect on cancer cells (REFERENCE).
PROCEDURE
E. coli TOP10 were transformed with two plasmids, one responsible for constitutive expression of LuxR transcription factor and one containing the azurin and sfGFP gene in the same operon under an AHL inducible promoter regulated by the activating complex of LuxR::AHL. A second transformant strain was created carrying the gene encoding for p28 instead of the complete azurin protein. Biological triplicates of the engineered bacteria were cultured in shaking flasks at small scale (5 mL), and different AHL induction conditions were applied to them once they reached in the exponential phase (OD600nm = 0.4), specifically:
- Induction with 10-4M of AHL
- Mock induction with DMSO
Additionally, bacteria transformed with the same plasmids lacking the Plux_azurin/gfp construct were also cultivated serving as negative control of azurin expression. The cultivation stopped 200 min after induction, a time point where gene expression seemed to have stopped according to separate characterization experiments (see here link to bfr). Samples from each culture were transfered in a 96-well plate and both their absorbanse at 600nm (Abs 600nm) and their sfGFP content was evaluated in a plate reader. In the same plate we also included several concentrations of purified sfGFP allowing us to create a standard curve of sfGFP concentration (Figure 1). This standard curve, eventually allowed us to quantify the expression of sfGFP in the culture samples (Figure 2). A detailed protocol is available in Protocols.
RESULTS
The mean sfGFP concentration per cell was plotted for the biological samples under examination (Figure 2).
![Standard curve of sfGFP concentration](https://static.igem.org/mediawiki/2017/8/85/T--ETH_Zurich--sfgfp_standard_curve.png)
![Assesing the expression of sfGFP from the azu/sfGFP and p28/sfGFP operon](https://static.igem.org/mediawiki/2017/3/3f/T--ETH_Zurich--azurin_p28-induction.png)
The overexression and accumulation of sfGFP only after AHL induction was also verified via:
- Fluorescent images of the cell cultures exposed to blue-light illuminator (Figure 3, top)
- Images of the bacterial pellets from the bacterial cultures (Figure 3, bottom)
![Pictures of AHL induced and non-induced cultures expressing azurin/sfGFP or p28/sfGFP](https://static.igem.org/mediawiki/2017/d/d6/T--ETH_Zurich--Wet_Lab_Experiments_Anti_Cancer_Toxin_fluorescent_normal_pic.png)
CONCLUSION
With this experiment we could indirectly verify the functionality of both the operons expressing azurin and p28 respectively. From this experiments we chose the clone 3 for the creation of azurin-rich and p28-rich bacterial lysates. Samples from these cultures were further analyzed via SDS-PAGE to verify the production of p28 and azurin in a direct way.
SDS-PAGE to Confirm AHL-Induced Expression of azurin and p28
OBJECTIVE
Fluorescence measurements showed indirectly the functionality of the operons expressing azu/sfGFP and p28/sfGFP. To directly confirm that bacterioferritin was indeed co-expressed, an SDS-PAGE analysis was performed.
PROCEDURE
Protein lysates were obtained from bacteria treated with different concentrations of AHL. To determine the concentrations of proteins, needed to prepare the samples for SDS-PAGE, Bradford protein assay was preformed prior to the SDS-PAGE analysis. A detailed protocol is available in Protocols.
RESULTS
To determine protein concentrations in the lysates via Bradford protein assay, a standard curve was first generated by using a protein standard, bovine serum albumin, and measuring absorbance of different dilutions of the standard. Second, absorbance of the unknown samples was measured and the results were fitted to the curve (Figure 4). Linkkkkk to Irma's
After determining the unknown concentrations, samples were prepared accordingly and subjected to SDS-PAGE analysis. Band sized approximately 14.8 kDa (which corresponds to azurin) were visible in the samples treated with AHL. No bands corresponding to the azurin-derived p28 peptide could be detected, mainly because of the its small molecular weight (27 aa, ~4 kDa). There were no bands in untreated samples or the negative control (Figure 5).
![](https://static.igem.org/mediawiki/2017/2/22/T--ETH_Zurich--_gel_azurin_p28_vasilis.png)
CONCLUSION
As expected, a band corresponding to azurin were visible in the sample induced with AHL. With this, to co-expression of azurin and GFP in the test strain for azurin-rich lysate production experiments was confirmed. The production of p28 from our engineered strain couldn't be confirmed and for this reason no further experiments were conducted with this strain.
Determining the optimal seeding cell concnetration for mamalian cell cultures
OBJECTIVE
With this experiment we wanted to define the optimal starting cell population of different cancer - cells lines, in order to avoid overgrowth and detachment of the mammalian cells in our experiments.
PROCEDURE
Variable amount of cells were seeded in a 96-well plate and their growth was assessed via microscopy after 24 hour growth in DMEM medium (supplemented with 1% streptomycin, 1% penicillin and 2% FBS).Two different cell - lines were tested, namely HEK-239 and HeLa CCL-2 lc. The use of HeLa CCL-2 lc allowed to monitor the growth of the cells via fluorescence microscopy as these cells are producing constituively citrine. A detailed protocol is available in Protocols.
RESULTS
From the microscopy pictures obtained after 24-hours incubation, the optimal cell concentration range was defined between 60'000 and 600'000 cells/mL in a 96-well plate. For higher concetrations the mamallian cell culture seemed to approach confluency.
CONCLUSION
The optimal seeding cell concentration range to avoid overgrowth was set to 100'000 cells/mL, a value within the range dictated by the experiment (60'000 and 600'000 cells/mL). The same value was used also in experiments were 1mL cultures were incubated in 12-well culture plates.
Assessing the Calcein AM staining assay: Killing Curve of Different Cell Lines with 5-Fluouracil
OBJECTIVE
With this experiment we wanted to assess the efficiency of the selected staining assay. Calcein AM peremeat dye, penetrates the cell membrane and upon interaction with active esterases in the mamalian cell cytosol, a fluorescent product is being generated, leading to the staining of living cells with active esterases in their cytosol. In this experiment, cell samples with diverse viability are generated by the application of a concetration gradient of a well-known cytotoxic agaent, namely 5-fluouracil REF. The goal is to create a killing curve were the fluorescent signal of the cell sample is dropping with increasing cytotoxic agent concentration.
PROCEDURE
HeLa CCL-2 cells were seeded in a 96-well plate with initial concentration of 100'000 cells/mL. After 24-hour growth the cells are incubated with the killing agent. After 24 hours the cells are, incubated with calcein AM solution and the viable fraction of the cells is assessed via fluorescnce microscopy (Figure 1) and on a spectrophotometer.
A third cell-line (HeLa CCL-2 lc) was also assessed for its viability, but without using calcein staining. The use of HeLa CCL-2 lc allowed the monitoring the growth of the cells via fluorescence microscopy and on a spectrophotometer as these cells are producing constitutively citrine. Decrease in cell viability would result in less cells per well due to the detachment of the apoptotic cells from the well. In terms of fluorescence taht would result in the decrease citrine related fluorescence. A detailed protocol is available in Protocols.
RESULTS
From the microscopy pictures obtained, we could verify the apoptotic effect on both the cell lines after 24-hours incubation with the killing agent (Figure 1).
CONCLUSION
The calcein AM assay could successfully distinguish between dead and alive cells under the selected experimental conditions. Moreover, the killing can also conveniently assessed in HeLa CCL-2 lc cells just by dead-cell removal and assessment of constitutively expressed citrine in the absence of any permeat dye.
Assessing the bacterial lysate tolerance of different cell lines
OBJECTIVE
With this experiment we wanted to assess the tolerance of different cell line upon incubation with different amount of azurin-free bacterial lysate. The information acquired from this expreriment helped us to define the amount of medium that can we substitute by our bacterial lysate without harming the cells neither because of bacterial toxins present in the lysate nor because of nutrient depravation.
PROCEDURE
For different cell-lines, namely, HeLa CCL-2, HeLa CCL-2 lc, HT-29 and HEK - 239 cells were seeded in 4 different 12-well culture plates with initial concentration of 100'000 cells/mL. After 24-hour growth the cells are incubated with increasing amounts of azurin-free lysate in order to assess the effect of the lysate on the cancer cell growth . Simultaneously, cell samples were also incubated with increasing amount of PBS, instead of lysate, in order to check the effect of nutrient depravation on the cell growth. After 24 hours the cells, apart from the HeLa CCL-2 lc, were incubated with calcein AM solution. The viability of the cells was assessed via fluorescnce microscopy (Figure 1). The samples of HeLa CCL-2 lc cell-line were direcly examined for their viability via fluorescence microscopy (HeLa CCL-2 lc) as these cells are producing constitutively citrine. A detailed protocol is available in Protocols.
RESULTS
From the microscopy pictures obtained, we could verify the apoptotic effect on both the cell lines after 24-hours incubation with the killing agent (Figure 1).
CONCLUSION
The calcein AM assay could successfully distinguish between dead and alive cells under the selected experimental conditions. Moreover, the killing can also conveniently assessed in HeLa CCL-2 lc cells just by dead-cell removal and assessment of constitutively expressed citrine in the absence of any permeat dye.
References
- write here