Team:UCL/Project/Marker

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Revision as of 20:00, 4 October 2013

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.

EXPERIMENTS AND RESULTS

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

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 mg/mL of Zeocin, our Biobrick is successful.

Characterization

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 bloated, 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 typhan 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.

Sample Average Diameter (um)
Healthy Cells 13.18
Non-transfected HeLa cells 16.85
Transfected HeLa cells 14.54

The non-transfected cells subjugated to zeocin clearly bloat, while the bloating in transfected cells is substantially less. Again, this is because She ble does not confer full zeocin immunity, but zeocin resistance (Oliva-Trastoy 2005). Visually, down the microscope, we observed that the in the control the cells would bloat 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.