Team:Glasgow/AzureA
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Azure A Staining
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Summary
Azure A (Dimethylthionine) is a blue dye of the Thiazin family which can be used to visually stain DNA down to quantities of 20ng. Azure A staining requires no expensive equipment or controlled disposal techniques like the common DNA stain Ethidium Bromide. Glasgow’s 2015 iGEM team utilised Azure A staining for the vast majority of the Agarose gel DNA purification/extractions we performed. We aim to promote Azure A staining in order to expand the participation of community and high school labs in iGEM and molecular biology, where they would not be able to meet the costs of equipment or disposal of Ethidium bromide for DNA visualisation.
Background
Azure A is produced when Methylene blue, another Thiazin stain, is oxidised. Methylene blue is less sensitive for staining DNA than its oxidation products, while Azure A demonstrates reduced background gel staining and a reduced required staining time (NCBE, 2003b). The Thiazin family is thought to stain nucleic acids through ionic interactions with the phosphate groups of the sugar-phosphate backbone (NCBE, 2003a); but weak intercalative interactions with DNA have also been described (Paul and Kumar, 2013).
The costs of staining DNA
Originally, the system containing UirS, UirR, and PlsiR accounts for a negative phototactic response to unidirectional UV-A light. The proposed mechanism puts UirS, a transmembrane protein of the CBCR family, as the molecule that perceives UV light. It is suggested that through a physical interaction between UirS and UirR and possibly a phosphotransfer from UirS to UirR, UirR is released from the transmembrane protein. The released UirR can now bind to DNA and UirR, which is similar to other activators of stress responses, was found to be a transcriptional activator of lsiR after binding to its promoter PlsiR .
Sigma-Aldrich sells 10ml of Ethidium bromide (EtBr) solution at a concentration of 10mg/ml for £45/$56 (E1510) (Sigma-Aldrich, 2015). We used a solution of 15ul (10mg/ml) EtBr per 0.5L of TAE running buffer for post-run staining, which works out to ~6666 0.5L stains per bottle of Ethidium. Traditional DNA staining with Ethidium bromide appears to be low-cost when only the cost of the stain is considered; with a cost per gel of £0.0067. However the cost to a lab is greater than simply the stock stain; Ethidium stained DNA requires visualisation on a UV-transilluminator, and the model of UV transilluminator available to us comes from a range which begins at £600/$900 (VWR, 2015). UV illumination requires an enclosed space where other people and sensitive items will not be damaged by irradiation. Additionally, imaging an Ethidium stained gel requires specialist camera filters to prevent camera sensor damage by the UV radiation, or the use of an enclosed illumination and photographing apparatus such as the BioRad Gel Doc™ XR system. Disposal of Ethidium bromide stained gels is also costly; EtBr has been found to be a potent mutagen in in vitro testing, thus disposal and handling is treated very seriously. The EtBr disposal policy of the University of Glasgow states that EtBr waste should be disposed of into biohazard marked containers, which must be uplifted by a 3rd party waste disposal service.
We have confirmed through the use of a laser scanner that PlsiR is not active when UirS and UirR are absent. PlsiR was ligated to GFP with two ribosome binding sites of different strength and no fluorescence was observed (the parts we used for this experiment were K1725401 and K1725402) (Chart 1). Moreover, cells that possess UirR but lack UirS also did not show levels of fluorescence above the expected for E. coli. Therefore, UirR is not sufficient to drive the activation of PlsiR.
Survivability
Introduction
The fact that UV exposure can be lethal to E.coli is well documented. After deciding to use a UV sensor to activate our system, it became obvious that we would have to examine the effects of exposing E.coli to the amount of UV needed to activate the sensor over time. To do this we generated a number of survival curves. (Please note, these experiments were not repeated enough to generate statistically significant results.) The graphs have been included as an indication of the thought process that went into guiding the experiments. Given more time we would have repeated these experiments many more times. Genotypes of the strains used can be found here for MG6115 TOP10 and DH5α and here for DS941.
Initial aims
Initially we wanted to explore what happened over a relatively small period of exposure, as our assumption was that we would see substantial reduction in a fixed number of E.coli, even over a relatively small time period. We also wanted to see how the different strains (DH5α ,TOP10) being used would respond compared to each other.
Since both DH5α and TOP10 are recA negative, and are therefore incapable of certain major kinds of DNA repair, we predicted that these strains would be more acutely affected than DS941 and MG6155.
Method
1ml 10x serial dilutions were made up to 10-6 from a 5ml overnight of each strain. 10µl spots of each dilution were then spotted onto LB agar plates.
These plates were then exposed to 50µmoles/m2/s of UVA at room temperature in illumination cabinets. Time points were then taken by removing plates from the illumination cabinet.
After illumination, plates were incubated at 37°C, under the assumption that every single viable cell will form a colony. The length of incubation is irrelevant, provided that every cell is given enough time to form a visible colony. This also forms the basis of our counting system, where a colony is assumed to have come from a single cell. We also make the assumption that cell division at the temperature and time we were running the experiment was negligible/nonexistent.
After incubation, the colonies on each spot/dilution were counted. The number of colonies from the lowest visible dilution (some dilutions formed a lawn of growth) were then multiplied by the dilution factor to approximate how many cells would be in 10µl of undiluted culture.
Results
Conclusion
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