Results

Overview

For a better understanding of the results of our project, we divided this part into three parts:

1. Experiments involving mammalian cells
2. Bacterial sensor
3. Chip

Experiments involving mammalian cells

Our first step needed to check the viability of our experimental set-up was to confirm the data found in the literature regarding the sensitivity to TRAIL and the elevated lactate production observed in cancer cells, but not in normal cells.

Sensitivity to TRAIL

Background

Apoptosis can be induced through a protein called TNF-related Apoptosis Inducing Ligand (TRAIL). TRAIL is a transmembrane protein which can be found also in solubilised form [de Bruyne et al, 2013 ] that recognizes the death receptors DR4, DR5, DCR1, and DCR2 [Johnstone et al, 2008] and it has been used in cancer treatment inducing the apoptosis of cancerous cells but with low cytotoxicity for healthy ones [Johnstone et al, 2008; Zhang et al, 2005).

Experiment overview

The first signal in our system is based on making cancer cells apoptotic while healthy cells remain untouched. Due to the properties of TRAIL explained before, we used it for a selective apoptosis induction in cancer cells.

The aim of this experiment was to check if cancer cells effectively undergo apoptosis after incubation with TRAIL. We also wanted to check which concentration of TRAIL have to be applied for best results and how long the incubation should last.

To test the predisposition of cancer cells to TRAIL-induced apoptosis we chose Jurkat cells, which are an immortalized human T lymphocyte cell line; and HL-60 cells, a line derived from acute myeloid leukemia. For a control with healthy cells we used a murine fibroblast cell line (3T3).

Methods and Results

Our first experiment consisted in incubating Jurkat and 3T3 cells with TRAIL for 4h and 6h (look protocol about apoptosis sensibility). Concentrations of 0, 50 and 100 ng/mL of TRAIL were used with each cell line. A positive control for Jurkat cells was applied by incubating these cells with PMA and ionomycin overnight. After that, FACS analysis was performed. To differentiate between apoptosis and necrosis, PI dye was used to stain necrotic cells. Apoptotic cells were detected by using Annexin V labelled with Alexa 488 (Life Technologies).

Unfortunately, despite doing several trials in which we incremented the number of hours for incubation (up to 48h) and tried different concentrations of TRAIL, we were not able to replicate the results we found in the literature. To see where the problem was, we decided to incubate the cells with 1000 ng/mL of TRAIL over a period of 48 h. If TRAIL was functional, we would expect to see the cells with a different morphology, which would mean that our detection system is not working even though apoptosis was properly induced. If TRAIL was not functional, we expect not to see morphological differences. After the 48h we saw that there were no differences in the morphology of the cells, which lead us to suppose that probably the sTRAIL we bought was not working as expected.

Our next step was to purchase another TRAIL called killerTRAIL (Enzo). To check whether this time our experiments were working, we added 100 ng/mL of TRAIL to Jurkat cells and measured the apoptosis at 2 h and 4 h. We observed that in 2 h apoptosis increased from about 2% of the population in non-treated samples to almost 70% in cells incubated with TRAIL. After 4 h the apoptotic cell number increased up to 75%. As the number of apoptotic cells is increasing slowly after 2 h, we decided to use this as our standard time for TRAIL incubation.

Figure 1. Jurkat cells with 0 and 100 ng/mL of TRAIL after 2 and 4 h. Apoptosis was detected in all cases with Annexin V (Q3).

Lactate production

Overview

A lot of cancer cells ungergo a metabolic transition, called Warburg effect, where they do the aerobic glycolysis to obtain ATP. A subproduct they generate in this kind of energy production id L-Lactate (Vander et al, 2009). Therefore, the excretion of lactate by cancer and healthy cells is different enough to be used as an indicator of cancer.

First, we wanted to check the how the production rates of lactate would vary in healthy and cancer cells. To this end, we analysed the medium in which our cells of interest had been incubated for a defined time in a defined seeding density. For detection of lactate in a sample, we used a L-lactate kit (Megazyme), in which two enzymatic reactions were performed. In the first, L-lactate and NAD&sup+; were converted into pyruvate and NADH+H&sup+; by L-lactate dehydrogenase. In the coupled reaction, pyruvate is used with D-glutamate by the D-glutamate-pyruvate transaminase to have the equilibrium of the first reaction in favour of the pyruvate. The concentration of NADH is finally measured as the optical density at 340 nm (OD340). The kit was used applying the provided protocol.

Results

The first problem we came across measuring the absorbance was the phenol red of the medium, which absorbs at the same wavelength as the lactate kit. Once the phenol red was removed, there was still another problem: a component of the medium seemed to react with our kit. After a bibliographic search, we decided that removing FBS from the medium was our best chance to get reliable calibration curves and measurements. We also incorporated a centrifugation step after the collection of the sample to remove any cell that could stay in suspension.

Co-culture of mammalian cells with bacteria

Overview

One issue that was concerning us was the possibility that the mammalian cells would behave in unusual ways in presence of bacteria. To exclude this possibility we decided to co-culture mammalian cells with bacteria for 3 h which is the time we expect that our test will last.

We used E. coli expresssing GFP and for the mammalian cell lines we used 3T3, HL-60 and Jurkat lines.

Results

We could observe that after the set time point the cells had the same morphology as in the control.

Bacterial sensor

INP-Annexin V expression

Overview

In Microbeacon system we rely on bacteria binding to cancer cells as a co-localization signal. The solution that we propose is to use Annexin V expressed on the bacterial membrane to bind cancer cells. Annexin V binds to phosphatidylserine, which is a membrane lipid usually found in the inner part of the cell, but in cells undergoing apoptosis it flips to the outer membrane due to the activity of flipases.

Annexin V is a soluble protein with an unknown function. As we want to express Annexin V in the outer membrane we need a protein that will export it. For this purpose we chose INP ((BBa_K523013)) and changed the YFP for a human Annexin V protein optimized for Escherichia coli codon usage (Life Technologies). We used a strong RBS (BBa_B0034), a low expression promoter (BBa_J23114) and a low copy plasmid pSEVA371 to avoid an excessive stress for the cell, as it is a membrane protein.

Results

To test our construct we used:

• J23114-B0032-INP-Annexin V in pSEVA371 (low copy plasmid)expressing RFP in TOP10 cells
• J23114 expressing RFP in TOP10 cells

To test whether our experiment works, we used for colonies with the above description. Then, as a first test, we did a test with beads. We used magnetic beads, which we incubated with antibodies for 30 min and then with the bacteria with different constructs. Unfortunately, we got no results from them. We also did a Western blot using our anti-Annexin V antibody (Affimetrix BMS147) observing no band in the Annexin V weight.

We wondered what could have gone wrong, so we checked with an RBS calculator (Salis Lab) whether the expression would be high enough with our design. There, we realized that Annexin V expression was too low and probably our protein was not being expressed. Using B0032 with INP-Annexin V we were getting 410 a.u., while if we changed the RBS to B0034, we would get 12 000 a.u. Therefore, we changed the RBS of INP-Annexin V and Annexin V to B0034. As can be seen in Figure X we can see a band at the expected 35.9 kDa. This confirms the expression of Annexin V in our cells.

Characterization of the lldR promoter

Overview

For the sensing of lactate inside our system we have a constitutive expression of LldR. LldR is a protein which binds to operator1 and operator2 and forms a loop, which does not allow the polymerase to synthesise the gene. Once lactate is added to our system, it sequester LldR and it cannot repress the promoter anymore, so LacI can be expressed.

An improvement to our system is the addition of LldP, a protein which is known to insert into the membrane and increase the importation rate of lactate into the cell.

To characterize the promoter, we put a gfp after it. Then, we measured a range of lactate concentrations in bacteria with:

1. a medium copy plasmid (pSEVA271) containing our designed lldR-operators with a promoter followed by a gfp.
2. the formed medium copy plasmid and a low copy plasmid (pSEVA371) containing lldR
3. the medium copy plasmid and a low copy plasmid containing lldR and lldP

Results

To find the optimal regulatory system, we designed nine versions of it:

Table X. Design of the promoter regulating the expression of LacI.

Characterization of the lacI-lldR promoter

Overview

Results

Quorum sensing modul

Overview

Results

Chip