Team:ETH Zurich/Results
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Results
Overview
For a better understanding of the results of our project, we divided it in three parts:
Experiments involving mammalian cells
Sensitivity to TRAIL
Overview
The first step in our design is to induce apoptosis in cancer cells. This can be done 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). Thanks to this property of TRAIL, we used it for a selective cancer apoptosis induction.
To test the predisposition of cancer cells to TRAIL we chose Jurkat cells, which are an immortalized human T lymphocytes cell line; and HL-60 cells, derived from acute myeloid leukemia. For a control with healthy cells we used a murine fibroblast cell line (3T3).
Results
Our first experiment consisted in incubating Jurkat and 3T3 cells with TRAIL for 4h and 6h (look protocol). Concentrations of 0, 50 and 100 ng/mL of TRAIL were used with each cell line. A positive control was incubated with PMA and ionomycin for Jurkat cells, which was incubated for 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 the concentration of TRAIL we could not replicate the results we found in the literature. This lead us to suppose that probably the sTRAIL we bought was not working as expected, as morphologically the cells also did not change.
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 passed from being about 2% of the population to almost 70%. After 4 h the apoptotic cell number increased up to a 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.
Lactate production
Overview
Cancer cells do the aerobic glycolysis to obtain ATP in a process called Warburg effect, and a subproduct they generate lactate (Vander et al, 2009). Therefore, the excretion of lactate by cancer and healthy cells is different enough to be used as a sensor.
First, we wanted to check the how the production rates of lactate would vary in healthy and cancer cells. For that, 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 with OD 340 nm. The kit was used using 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 that the lactate kit. Once the phenol red was removed, there was still another problem: the medium was producing a reaction. After a bibliographic search, we decided that removing FBS from the medium was our best choice. We also incorporated a centrifugation step after the collection of the sample to remove any cell that could stay in suspension.
As we expected, the lactate concentration in our cancer lines (HL-60 and Jurkat cells) was higher than in the control 3T3 cell line (Figure X), a 5 fold-change between the HL-60 and the 3T3 cells and a 6.7 fold change between Jurkats and 3T3.
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 kidnaps 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:
- a medium copy plasmid (pSEVA271) containing our designed lldR-operators with a promoter followed by a gfp.
- the formed medium copy plasmid and a low copy plasmid (pSEVA371) containing lldR
- 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.
| O1-promoter-O2 | promoter-O1-O2 | promoter-O1-spacer-O2 | |
| J23100 | p69 | p63 | - |
| J23117 | p70 | p65 | p66 |
| J23118 | p71 | p67 | p68 |
| plldR | p62 | - | - |