Tests

Damage tolerance assay

To measure the DNA damage tolerance conferred by each part, we used Mitomycin C and Hydrogen peroxide as a source of DNA damage and then assayed the survival rates. Transformed E. coli cells were plated on agar plates at different dilutions, air dried, and then exposed to different doses of UV radiation. Plates were wrapped with aluminum foil and incubated in the dark. Colony-forming units were scored after 16h incubation at 37°C. For detailed protocols, refer to the Protocols page.

The results will be up soon!

SOS promoter assay

It is very important to be able to regulate gene expression as you like in biotechnology. To apply a regulator, such as a promoter, of the relevant strength to a gene, it is first essential to take an accurate measurement of the activity of regulators. However, it is difficult to measure the activity of regulators because the activity often changes depending on the conditions. In order to deal with this problem, Kelly et. al. (2009) proposed a BioBrick promoter assay system based on relative promoter strengths. Their system measures the fluorescence intensity of GFP placed downstream of the promoter of interest, which is used to compute the absolute promoter strength. This value is normalized to the strength of a “standard” promoter which is simultaneously measured so that differences between experiments could be minimized. In this project, we suggest an alternative promoter assay system. In the dual luciferase assay(Hannah et al 1998), the luminescent intensity caused by firefly luciferase, used as a reporter of the promoter of choice, is compared to that caused by renilla luciferase, used as a reporter for a control promoter, both placed within the same plasmid. Importantly, because the promoter to be measured and its comparison are placed within the same plasmid, differences in expression between cells are minimized. In addition, the need to normalize cell densities between sample and control is abolished, which can often be a difficult and inaccurate task. Also, the use of luciferase as a reporter allows a quick and accurate measurement and in addition is not fluorescence-based, nullifying the need to worry about fluorescence quenching.

The results will be up soon!