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Team:Bologna/Wetlab
From 2008.igem.org
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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.
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 2-3)
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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 .
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The matrix is obtained through the pressure of the cover part over the mold part
and that is the result:
