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Team:Bologna/Results
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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.
UV Side Effect
In addiction to the germicidal effect UV radiation can also cause erythema (reddening of the skin)
and conjunctivitis (inflammation of the mucous membranes of the eye). Because of this,exist limit
to the exposure[1]that depends by the irradiance of the ultraviolet- lamps(figure 4-5).
Homemade UV transilluminator
When this device are used, it is important to design systems to exclude UV-C leakage and so avoid figure 4: Ultraviolet treshold limited value figure 5: Permissible ultraviolet exposure these effects.The UV source that have been used in this project is a GW6 Sylvania, a lamp that emitt UVC at 253,7nm near the absorption's peak of DNA with an optical output power of 1,6W; to use it safely we built a box of mdf(medium density-fibreboard) that surrounds the lamp and using a diaphragm that allows passage only to the light absorbing the remainder. Moreover we insert in that box two pierced brackets, those permits to choose the distance between the lamp and the sample and then in accord with the Lambert-Beer law's the exposure time.
Finally we embedded this structure in a polistyrene box for its handling and greater safety.(figure 6)
Gel matrix
Our project use UV light for its space selectivity, that gives the possibility to irradiate a target zone without interfering with the other. To do that we build a mold to make a matrix of agarose gel;this give us a square of 25mm side's with 16 cells inside where we can locate bacteria.
Once do that is easy with an optical mask, like those used in photolithography for electronics
circuits, stimulate only the selected bacteria.To realize this device we use palsticard because it is
very easy to shape and clean.
The mold is made of two parts that will form a sandwich with the slide: one will create the shape
of the gel matrix the other is a cover that that will give the picture into gel .
The matrix is obtained through the pressure of the cover part over the mold part
and that is the result:
