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 (OD_600nm = 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).

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)

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.

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).

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.

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.

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.