Team:UCL/Project/Marker

ZEOCIN RESISTANCE

For Selecting Transfected Cells

To easily select cells that were transfected with our genetic circuit, we required a selectable marker that would work in all of our chassis, particularly HeLa cells and microglia, and would enable us to easily eliminate cells that have not taken up our recombinant plasmid. Zeocin is a widely used glycopeptide antibiotic, a formulation of phleomycin D1. It is capable of binding to and cleaving DNA, leading to cell necrosis in both eukaryotes and aerobic prokaryotes. Commonly outside of cells, in copper-chelated form, zeocin is inactive. When zeocin enters a cell, the Cu2+, which makes it appear blue, is reduced to Cu+ and then removed, activating zeocin, which then intercalates into DNA (Invitrogen).

A 375 base pair bacterial gene encodes the Streptoalloteichus hindustanus bleomycin resistance protein (She ble protein). The She ble protein in mammalian cells is predominantly localised at the nucleus, specifically at euchromatin (Calmels et al. 199). This small protein that has a strong affinity for antibiotics on a one to one ratio. It prevents zeocin from being activated by ferrous ions and oxygen, meaning it cannot react in vitro with DNA. However, the protection it confers, in human cells at least, while considerable, is not complete. However, it is an extremely useful selectable marker, that will be invaluable to the iGEM registry (Oliva-Trastoy 2005).

In order to establish that this BioBrick worked, we had to first determine Zeocin’s killing efficacy against HeLa cells by creating a kill curve.

Creating The BioBrick

In order for mammalian cells to express Zeocin resistance, our Zeocin resistance biobrick (BBa_K1018001) includes a CMV promoter.

This biobrick benefits the iGEM Registry tremendously by providing a suitable selectable marker for cell culture and mammalian transfection, which was previously non-existent in the iGEM Registry. This biobrick will make mammalian transfection easier and will encourage iGEM teams to venture more into mammalian synthetic biology.

Growth Curve

Before using HeLa cells for transfection and characterisation, we carried out basic characterisation of the chassis. For this, we conducted a HeLa growth curve.

There is an exponential growth until the 4th day. After the 4th day, the growth of HeLa cells slows down. Some cells start to detach and die from over-confluency.

Through this graph, we were able to decide that we would split or passage HeLa cells every 3 to 4 days to maintain good cell health. Maintaining good cell confluence is important for maximum transfection efficiency.

The ideal HeLa confluency for transfection is 70%. Hence, from this graph, we can plan to split the cells one day before transfection to attain 70% confluency.

Zeocin Kill Curve

In order to determine the concentrations of Zeocin at which HeLa cells start to die, we carried out a Zeocin Kill curve.

This helped us to decide the concentrations of Zeocin we would use to characterise our Zeocin resistance Biobrick (BBa_K1018001).

From this data, we hypothesised that if our HeLa cells survive in 50-200 ug/mL of Zeocin, our Biobrick is successful.

By carrying out this Kill Curve, we were able to observe the appearance of healthy cells versus swollen and dead cells for objective characterisation of our Zeocin resistance biobrick.

Characterisation

In order to characterise our new BioBrick, we seeded the wells of a six-well plate with HeLa cells at passage 20 and transfected all but one (the control) of these wells with (BBa_K1018001) using SuperFect transfection reagent. We used three six-well plates, exposed one to 50 ug/ml zeocin, another to 100 ug/ml zeocin and the last to 150 ug/ml Zeocin. These concentrations were chosen based on our kill curve data. We plotted the percentage of cells we deemed viable in our plates in a blind study over eight days. Viability was assessed visually by noting the surface area occupied by bloated/dead cells versus healthier cells.

Our graphs clearly demonstrate that the transfected HeLa wells fared better than the non-transfected ones, by a margin of about 10%. This is to be expected, for three reasons:

Firstly, the sample of (BBa_K1018001) was impure and so not all of the transfected cells will have been transfected with She ble. Moreover, we expect only about 40% of the cells to have been transfected at all. This is a conservative estimate based on the fact that we have not worked with mammalian cells or done a transfection before iGEM.

Secondly, viability readings are distorted by the fact that HeLa cells die due to over confluence (population stress and lack of nutrients) meaning that, especially in the transfected wells, not all cell death was due to zeocin action. For more information, see the box below.

Thirdly, the she ble gene does not confer full immunity to transfected cells, only a degree of resistance, which is why the transfected cells die quicker at higher zeocin concentrations. All of the graphs show the transfected cells experiencing a slight rise in viability at the 6-7th day. We hypothesise that this is because zeocin resistant cell clusters expand into the space left by unsuccessfully transfected HeLa cells that have died. The curve continues its downward trend, however, because of over confluence.

Qualitative Data

There is steady slow growth for all wells up until the 100th hour of Zeocin exposure. After the 100th hour, Zeocin starts to take effect and the HeLa cells start to decrease in confluency, indicating gradual cell death. The Control well decreases in confluency steadily over time. Well 3 continues to grow, indicating that most HeLa cells in the well have been successfully transfected.

Comparing the experimental wells and the control well, there is an obvious difference in growth pattern and confluency as exposure time to Zeocin increases. By the 200th hour, the confluency difference between experimental wells and control well differ by 20-60%.

Types of Cell Death

Above. Cell death due to over confluence in our HeLa cells transfected with (BBa_K1018001) in 200 ug/ml of Zeocin, after four days.

Below. Cell death due to zeocin in our non-transfected HeLa cells in 200 ug/ml of zeocin, after four days.

In both our transfected cell wells and in the control there is net cell death over time. Though it is clear from the above graphs that the transfected cells fair better over time, it is important to note that the cell death these wells sustained was proportionally more due to over confluence than due to Zeocin. We ran a set of transfected wells and a control of non transfected HeLa cells over three days, and took a set of images, two of which are shown to the right. The upper image shows an over confluent dish of cells, in which the viability is low because cells are dying due to population stress and lack of nutrients. The swollen, stretched, spindly HeLa cells in the lower image are characteristic of Zeocin imposed cell death; they have lysed or are ready to lyse.

To show this quantitatively, we used a Vi-Cell (cell viability analyser), which identifies dead cells using tryphan blue and measures cell diameter, to take readings from a sample of healthy cells, our control cells at 200 ug/ml Zeocin and a well of transfected cells at 200 ug/ml Zeocin.

The non-transfected cells susceptible to Zeocin are clearly swollen, while the swelling in transfected cells is substantially less. Again, this is because the she ble gene does not confer full Zeocin immunity, but Zeocin resistance (Oliva-Trastoy 2005). Visually, down the microscope, we observed that the 'control' cells would swell to up to about 20 um in diameter, small clusters of smaller diameter, healthier cells. These clusters were more common in the transfected HeLa wells, assumedly where surviving transfected cells have proliferated into gaps left by their non-transfected neighbours dying.

The photo to the right clearly shows that the control has less viable cells than the transfected well. This photo is of the 150 ug/ml zeocin at day eight. The growth medium in the control is far pinker than in the other wells. This is because live cells use of nutrients in the growth medium and sap its colour.