Team:UCL/Project/Marker

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Revision as of 17:05, 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