Revision as of 09:40, 29 October 2008

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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.

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(next figure).

Ultraviolet treshold limited value

Permissible ultraviolet exposure

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.

mdf box structure

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:

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 and that is the result:
 [[Image:imm3.jpg|center|thumbnail|450 px]]
-==Protocol used for UV induction==
-The plasmid with our construct has been transformed in DH5alfa bacteria by standard protocol and one colony from the plate has been picked and cultured overnight in  5ml LB medium broth with ampicilline.
-The day after the culture  has been diluted in 5ml LB and antibiotic in order to have 0.1 starting OD bacteria culture and let it grown for one hour; after that the culture has been divided in five falcons with 1ml of bacteria, ready to be induced.
-Using 1ml of culture is a choice done in order to have a thickness as thick as possible to perform an irradiation as uniform as possible .
-Tests with difference distances from the lamp with different exposition times were done in order to respect the maximum lethal UV dose of bacteria and avoid mutagenesis factors in the cell.
-The following table illustrates the tests setup used:
-[[Image:tabuv.jpg|center]]
-After induction by UV the samples were kept in dark by silver paper to increase the RecA and LexA answer and let grown for 2h.
-The OD sample is measured and the sample transferred in a 1ml eppendorf spinned at 6000-8000rpm for three minutes and  pellet collected in order to measure fluorescence level by microscope and Bacteria Visual Fluo Software.
-Unfortunately, using Falcon tube and not the correct time/distance induction the experimental tests showed weak GFP expression using the construct with straighter promoter. It could be depended by the low sensibility of SOS system with our setup tests and not uniform irradiation of the sample. In Fig.3 is possible to see a leak answer by bacteria stimulated by UV at the distance of 4cm with one second time exposure.
-[[Image:colonia1.jpg|400px|thumbnail|Fig.3|center]]
-In order to show the correct functionality of our construction, induction by peroxide hydrogen was used, in fact a low concentration of H_2 O_2 (1-3mM) results in SOS gene induction in wild-type cells ''[6], [7]''.
-The induction of SOS by H_2 O_2 is an important event, since RecA and recBC mutant cells are very sensitive to H_2 O_2 treatment probably due to the lack of recombinational repair necessary for the H_2 O_2 – induced lesions ''[8]''.
-<br><br>
-[[Image:schema.jpg|right]]
-The reaction of hydrogen peroxide with transition metals imposes on cells an oxidative stress condition that can result in damage to cell components such as proteins, lipids and principally to DNA. Escherichia Coli cells are able to deal with these adverse events via DNA repair mechanisms or OxyR and SosRS anti-oxidant inducible pathways which are elicited by cells to avoid the introduction of oxidative lesions by hydrogen peroxide.
-Among the systems the OxyR gene interacts with, there is the SOS response.
-<br><br><br><br><br><br><br><br><br>
-= Single operator isolation =

Team:Bologna/Wetlab

From 2008.igem.org

Revision as of 09:40, 29 October 2008

Contents

LAB Experiment

UV Side Effect

Homemade UV transilluminator

Gel matrix