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Team:Bologna/UV radiation/
From 2008.igem.org
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UV Spectrum
Ultraviolet is that part of electromagnetic radiation bounded by the lower wavelength extreme of the visible spectrum and the upper end of the X-ray radiation band. The spectral range of ultraviolet radiation is, by definition, between 100 and 400 nm and is invisible to human eyes. The UV spectrum is subdivided into three bands: UV-A (long-wave) from 315 to 400 nm, UV-B (medium-wave) from 280 to 315 nm, UV-C (short-wave) from 100 to 280 nm. A strong germicidal effect is provided by the radiation in the short-wave UV-C band.
E.coli SOS System
The maximum UV germicidal effect coincides with the peak absorbance of DNA (near 260nm) due to the dimerization of two adjacent thymines. That can be seen in the Fig.1 where is showed the living population of bacteria after irradiation.
E.Coli cells have a system that recovery DNA damage when it occurs and the best studied transcriptional response to DNA damage is the SOS response [Friedberg et al., 1995; Walker, 1996].
This systems can be divided into two class: the SOS Photoreactivation repair and the SOS triggered by RecA protein. The first uses the photolyase, a poorly expressed enzyme(encoded by genes phrA and phrB) which binds the pyrimidine dimers and uses the blue light to split them apart as showed in Fig.2.
Instead single stranded DNA produced by several DNA-damaging agents can be bound by RecA protein, resulting in conversion of this protein to its activated form. The RecA repair system doesn’t need light and Lexa protein controls the expression of 43 genes [Courcelle et al. (2001)] that cooperate together to repair the extensive genetic damage. RecA and LexA proteins play an important rule on the regulatory of SOS Recombination System.
A LexA binding site is present in all the SOS promoters genes' and it works as a repressor of SOS system. In presence of DNA damage (DNA Single Strains) RecA becomes active and interacts with LexA protein , the repressor of the SOS genes [Wagner et al., 1999]. This interaction triggers the autocatalytic cleavage of LexA and consequent destruction of its ability to function as a repressor, which results in the derepression of SOS genes (Mustard and Little, 2000; Fernandez De Henestrosa et al., 2000).
When the damage is repaired, DNA single strains are not present in the cell and the RecA protein no longer promotes the auto-cleavage of the LexA which is restored to its initial repression level.
LAB Experiment
In our genetic bi-stable, the amount of molecules produced to switch the system from the two possible steady state is ruled by LacI and TetR protein. The production of LacI molecules by UV induction can be tested replacing the LacI gene by GFP, so it is possible to have a relation of the strength of LacI synthesis measuring the value of its fluorescence. BBa_K079049 and BBa_K079050 are two new construct submitted to the registry by Bologna’s Igem Team 2008. These UV test circuits can be presented schematically by the following sequence:
- Promoter Constitutive Family: BBa_J23118 (1429 strength ) or BBa_23100 (2547 strength);
- LexA Operator Site Binding (K079040 ) ;
- Rbs with Green fluorescence Protein LVA and Terminator (BBa_I763020)
In order to generate the induction by Uv, a box with UV lamp was realized.
