Part:BBa_K1018001:Design
she ble gene conferring Zeocin resistance
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 410
Illegal NgoMIV site found at 1442
Illegal NgoMIV site found at 1503
Illegal NgoMIV site found at 1617 - 1000COMPATIBLE WITH RFC[1000]
Design Notes
Description
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.
This biobrick benefits the iGEM Registry tremendously by providing a suitable selectable marker for cell culture and mammalian transfection.
Characterisation
To characterise this biobrick, we grew HeLa cells in 50 ug/ml, 100 ug/ml and 150 ug/ml concentrations of Zeocin.
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, becaase of over confluence.
Cell death due to over confluence in our HeLa cells transfected with BBa_K1018001 in 200 ug/ml of Zeocin, after four days.
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.
Sample Average Diameter (um) Healthy Cells 13.18 Non-transfected HeLa cells 16.85 Transfected HeLa cells 14.54
The non-transfected cells susceptible to Zeocin clearly swollen, while the swelling in transfected cells is substantially less. Again, this is because the she ble does not confer full Zeocin immunity, but Zeocin resistance [1]. Visually, down the microscope, we observed that the in the control the 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.
Source
Streptoalloteichus hindustanus
References
"Resistance to the antibiotic Zeocin by stable expression of the Sh ble gene does not fully suppress Zeocin-induced DNA cleavage in human cells.(Trastoy, Manel Oliva (2005) Nov;20(6):467.)