http://2011.igem.org/wiki/index.php?title=Special:Contributions&feed=atom&limit=50&target=Shao2011.igem.org - User contributions [en]2024-03-29T05:03:06ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/File:Combined_parts_effect.pngFile:Combined parts effect.png2011-11-04T23:29:52Z<p>Shao: </p>
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<div></div>Shaohttp://2011.igem.org/File:IPTG_induction_effects.pngFile:IPTG induction effects.png2011-11-04T23:29:49Z<p>Shao: </p>
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<div></div>Shaohttp://2011.igem.org/File:Viability_mitomycin.pngFile:Viability mitomycin.png2011-11-04T23:29:45Z<p>Shao: </p>
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<div></div>Shaohttp://2011.igem.org/Team:Osaka/ProtocolsTeam:Osaka/Protocols2011-10-28T23:41:04Z<p>Shao: /* SOS promoter assay 2: GFP */</p>
<hr />
<div>{{Osaka}}<br />
<div class="padding"><br />
== Protocols ==<br />
=== Cell survival assay 1: UV irradiation ===<br />
#Pre-culture transformed cells in 3ml of LB medium at 37&deg;C for 16h.<br />
#Induce parts with IPTG addition (to final concentration of 100&micro;M) for 1h.<br />
#Dilute pre-culture samples according to intended UV energy dosage (if dosage is high, lower dilution rate is needed as fewer cells are expected to survive).<br />
#Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.<br />
#Irradiate cells on the agar with UV light at desired energy dosage.<br />
#Wrap plates in aluminium foil and incubate at 37&deg;C.<br />
#After 16h, count number of colonies formed on control (non-irradiated) and UV-irradiated plates.<br />
<br />
<br />
=== Cell survival assay 2: Mitomycin C ===<br />
#Pre-culture transformed cells in 3ml of LB medium at 37&deg;C for 16h.<br />
#Induce parts with IPTG addition to final concentration of 100&micro;M and incubate for 1h.<br />
#Add mitomycin C to desired final concentration (we used 2&micro;g/ml) and incubate at 37&deg;C for a further 2h.<br />
#Centrifuge, discard mitomycin C-spiked medium and resuspend with fresh LB medium.<br />
#[RECOMMENDED] Dilute pre-culture samples according to expected survival rate, as determined from a preliminary experiment (lower dilution rate is needed if fewer cells are expected to survive).<br />
#Pipette 50μl to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness. <br />
#Wrap plates in aluminium foil and incubate at 37&deg;C.<br />
#After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates.<br />
<br />
<br />
=== SOS promoter assay 1: Carotenoid biosynthesis ===<br />
#Pre-culture transformed cells in 8ml of LB medium at 37&deg;C for 12h.<br />
#Transfer pre-culture to OD600 measurement dish (&oslash;50mm).<br />
#Irradiate with UV light at desired energy dosage.<br />
#Incubate irradiated cells for a further 2h.<br />
#Measure OD600 as a surrogate for cell density.<br />
#Transfer cells into 15ml Falcon tubes and centrifuge.<br />
#Discard supernatant, add 1ml water to wash cells.<br />
#Repeat centrifugation and decantation.<br />
#Add 500μl acetone and vortex.<br />
#Incubate at 55&deg;C for 15min.<br />
#Measure absorbance at 474nm. Use 100% acetone as blanks, and divide absorbances by OD600 values.<br />
<br />
<br />
=== SOS promoter assay 2: GFP ===<br />
#Pre-culture transformed cells in 20ml of LB medium at 37&deg;C for 12h.<br />
#Transfer pre-culture to OD600 measurement dish (&oslash;50mm).<br />
#Irradiate with UV light at desired energy dosage.<br />
#Incubate irradiated cells for a further 2h.<br />
#Measure OD600 as a surrogate for cell density.<br />
#Transfer cells into 50ml Falcon tubes and centrifuge.<br />
#Discard supernatant, add 4ml water to wash cells.<br />
#Repeat centrifugation and decantation.<br />
#Resuspend cells in 4ml water.<br />
#Measure fluorescence at 395nm excitation and 509nm emission. Use pure water as blanks, and divide emission values by OD600 values.<br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProtocolsTeam:Osaka/Protocols2011-10-28T23:33:18Z<p>Shao: /* SOS promoter assay 2: GFP */</p>
<hr />
<div>{{Osaka}}<br />
<div class="padding"><br />
== Protocols ==<br />
=== Cell survival assay 1: UV irradiation ===<br />
#Pre-culture transformed cells in 3ml of LB medium at 37&deg;C for 16h.<br />
#Induce parts with IPTG addition (to final concentration of 100&micro;M) for 1h.<br />
#Dilute pre-culture samples according to intended UV energy dosage (if dosage is high, lower dilution rate is needed as fewer cells are expected to survive).<br />
#Transfer 50μl of diluted pre-culture onto LB agar plates and spread evenly. Air-dry the plates to remove excess wetness.<br />
#Irradiate cells on the agar with UV light at desired energy dosage.<br />
#Wrap plates in aluminium foil and incubate at 37&deg;C.<br />
#After 16h, count number of colonies formed on control (non-irradiated) and UV-irradiated plates.<br />
<br />
<br />
=== Cell survival assay 2: Mitomycin C ===<br />
#Pre-culture transformed cells in 3ml of LB medium at 37&deg;C for 16h.<br />
#Induce parts with IPTG addition to final concentration of 100&micro;M and incubate for 1h.<br />
#Add mitomycin C to desired final concentration (we used 2&micro;g/ml) and incubate at 37&deg;C for a further 2h.<br />
#Centrifuge, discard mitomycin C-spiked medium and resuspend with fresh LB medium.<br />
#[RECOMMENDED] Dilute pre-culture samples according to expected survival rate, as determined from a preliminary experiment (lower dilution rate is needed if fewer cells are expected to survive).<br />
#Pipette 50μl to LB agar plates and spread evenly. Air-dry the plates to remove excess wetness. <br />
#Wrap plates in aluminium foil and incubate at 37&deg;C.<br />
#After 16h, count number of colonies formed on control and mitomycin C-treated inoculum plates.<br />
<br />
<br />
=== SOS promoter assay 1: Carotenoid biosynthesis ===<br />
#Pre-culture transformed cells in 8ml of LB medium at 37&deg;C for 12h.<br />
#Transfer pre-culture to OD600 measurement dish (&oslash;50mm).<br />
#Irradiate with UV light at desired energy dosage.<br />
#Incubate irradiated cells for a further 2h.<br />
#Measure OD600 as a surrogate for cell density.<br />
#Transfer cells into 15ml Falcon tubes and centrifuge.<br />
#Discard supernatant, add 1ml water to wash cells.<br />
#Repeat centrifugation and decantation.<br />
#Add 500μl acetone and vortex.<br />
#Incubate at 55&deg;C for 15min.<br />
#Measure absorbance at 474nm. Use 100% acetone as blanks, and divide absorbances by OD600 values.<br />
<br />
<br />
=== SOS promoter assay 2: GFP ===<br />
#Pre-culture transformed cells in 20ml of LB medium at 37&deg;C for 12h.<br />
#Transfer pre-culture to OD600 measurement dish (&oslash;50mm).<br />
#Irradiate with UV light at desired energy dosage.<br />
#Incubate irradiated cells for a further 2h.<br />
#Measure OD600 as a surrogate for cell density.<br />
#Transfer cells into 50ml Falcon tubes and centrifuge.<br />
#Discard supernatant, add 4ml water to wash cells.<br />
#Repeat centrifugation and decantation.<br />
#Resuspend cells in 2ml water.<br />
#Measure fluorescence at 395nm excitation and 509nm emission. Use pure water as blanks, and divide emission values by OD600 values.<br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T21:22:36Z<p>Shao: /* Tests */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
=== Damage tolerance assay ===<br />
To measure the DNA damage tolerance conferred by each part, we used UV irradiation 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&deg;C. For detailed protocols, refer to the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].<br />
<br />
The tolerance parts tested were as follows:<br />
=====Parts containing one gene each=====<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
*CDS: PprI, PprA, PprM or RecA<br />
<br />
=====Parts containing two genes=====<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
*CDS1+2: PprI+RecA, PprA+RecA, PprM+RecA, PprI+PprA, PprI+PprM, PprA+PprM<br />
<br />
Previously we tested some of these parts without IPTG induction. With the rationale that IPTG induction may increase expression of the protective proteins, we repeated past characterization tests with the inclusion of IPTG addition, and obtained slightly different results. These are indicated in the chart below.<br />
<br />
[[File:2011_osaka_tolerance_results.png|800px]]<br />
<br />
==== Discussion ====<br />
===== Single-gene parts =====<br />
*As before, PprI did not appear to significantly increase tolerance, corroborating with its known role as an inducer of other radiotolerance proteins. As ''E. coli'' would lack these required ''D. radiodurans'' proteins it is expected that PprI would not be able to confer tolerance on its own.<br />
*On the other hand, PprA, once induced with IPTG, seemed to confer a degree of DNA damage tolerance to ''E. coli''. This is not surprising considering PprA's function as a direct executor of DNA damage repair.<br />
*''D. radiodurans'' RecA was indicated in our previous experimental results to confer the highest tolerance among the four radiotolerance genes; however, here, its effect was less than that of either PprA or PprM. This might be because it plays a complementary role to native ''E. coli'' RecA, thus the higher expression level induced by IPTG was of no additional benefit.<br />
*Perhaps the most unexpected result was that from PprM. In our previous experiment (using non-induced parts), PprM also appeared to confer a degree of tolerance although it was not significant enough to be of note. Here, IPTG-induced PprM levels seem to provide a significant amount of DNA damage tolerance. This is somewhat in contradiction to literature which describes PprM as a modulator of the PprI-dependent damage response that depends on downstream effector proteins (PprA etc) to carry out its protective role.<br />
<br />
===== Two-gene combinations =====<br />
*While PprI alone did not confer any tolerance, the combination of PprI and PprA worked to some degree. This is in accordance with the role of PprI as an inducer of PprA. What was interesting is that, PprA alone appeared to confer even higher tolerance. Perhaps expression of PprI is somehow detrimental to the host ''E. coli'' cells.<br />
*The combination of PprI and RecA produced high level of tolerance, which agrees with the role of PprI as an inducer of ''D. radiodurans'' RecA function.<br />
*On the other hand, while literature mentions that PprM is not a modulator of RecA, here we see a significant increase in tolerance when these two genes are coupled. It could be explained by the fact that PprM is known to induce/modulate other, unknown proteins and some of these proteins may have homologs in ''E. coli'' that benefit from the presence of PprM.<br />
<br />
===== Conclusion =====<br />
We have obtained several interesting results from our DNA damage tolerance assays. First and foremost, PprM appears to confer a significant degree of tolerance to ''E. coli'', both on its own and in combination with other genes. Perhaps its role as a mere modulator of the PprI-dependent DNA damage response needs to be revised, or perhaps it is capable of regulating certain ''E. coli'' genes to advantageous effect.<br />
<br />
In addition, our results indicated that PprI, as a global regulator of the DNA repair system, does not confer tolerance to ''E. coli''. This is in contradiction to a previous report that PprI confers radiotolerance to ''E. coli'' cells. Perhaps codon optimization and a better expression system are needed to make our PprI BioBrick functional.<br />
<br />
Finally, we have shown that PprA, when expressed in sufficient quantities, does appear to confer tolerance to ''E. coli''.<br />
<br />
<br />
<br />
=== SOS promoter assay ===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
<br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T21:21:34Z<p>Shao: /* Single-gene parts */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
=== Damage Tolerance ===<br />
To measure the DNA damage tolerance conferred by each part, we used UV irradiation 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&deg;C. For detailed protocols, refer to the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].<br />
<br />
The tolerance parts tested were as follows:<br />
=====Parts containing one gene each=====<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
*CDS: PprI, PprA, PprM or RecA<br />
<br />
=====Parts containing two genes=====<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
*CDS1+2: PprI+RecA, PprA+RecA, PprM+RecA, PprI+PprA, PprI+PprM, PprA+PprM<br />
<br />
Previously we tested some of these parts without IPTG induction. With the rationale that IPTG induction may increase expression of the protective proteins, we repeated past characterization tests with the inclusion of IPTG addition, and obtained slightly different results. These are indicated in the chart below.<br />
<br />
[[File:2011_osaka_tolerance_results.png|800px]]<br />
<br />
==== Discussion ====<br />
===== Single-gene parts =====<br />
*As before, PprI did not appear to significantly increase tolerance, corroborating with its known role as an inducer of other radiotolerance proteins. As ''E. coli'' would lack these required ''D. radiodurans'' proteins it is expected that PprI would not be able to confer tolerance on its own.<br />
*On the other hand, PprA, once induced with IPTG, seemed to confer a degree of DNA damage tolerance to ''E. coli''. This is not surprising considering PprA's function as a direct executor of DNA damage repair.<br />
*''D. radiodurans'' RecA was indicated in our previous experimental results to confer the highest tolerance among the four radiotolerance genes; however, here, its effect was less than that of either PprA or PprM. This might be because it plays a complementary role to native ''E. coli'' RecA, thus the higher expression level induced by IPTG was of no additional benefit.<br />
*Perhaps the most unexpected result was that from PprM. In our previous experiment (using non-induced parts), PprM also appeared to confer a degree of tolerance although it was not significant enough to be of note. Here, IPTG-induced PprM levels seem to provide a significant amount of DNA damage tolerance. This is somewhat in contradiction to literature which describes PprM as a modulator of the PprI-dependent damage response that depends on downstream effector proteins (PprA etc) to carry out its protective role.<br />
<br />
===== Two-gene combinations =====<br />
*While PprI alone did not confer any tolerance, the combination of PprI and PprA worked to some degree. This is in accordance with the role of PprI as an inducer of PprA. What was interesting is that, PprA alone appeared to confer even higher tolerance. Perhaps expression of PprI is somehow detrimental to the host ''E. coli'' cells.<br />
*The combination of PprI and RecA produced high level of tolerance, which agrees with the role of PprI as an inducer of ''D. radiodurans'' RecA function.<br />
*On the other hand, while literature mentions that PprM is not a modulator of RecA, here we see a significant increase in tolerance when these two genes are coupled. It could be explained by the fact that PprM is known to induce/modulate other, unknown proteins and some of these proteins may have homologs in ''E. coli'' that benefit from the presence of PprM.<br />
<br />
===== Conclusion =====<br />
We have obtained several interesting results from our DNA damage tolerance assays. First and foremost, PprM appears to confer a significant degree of tolerance to ''E. coli'', both on its own and in combination with other genes. Perhaps its role as a mere modulator of the PprI-dependent DNA damage response needs to be revised, or perhaps it is capable of regulating certain ''E. coli'' genes to advantageous effect.<br />
<br />
In addition, our results indicated that PprI, as a global regulator of the DNA repair system, does not confer tolerance to ''E. coli''. This is in contradiction to a previous report that PprI confers radiotolerance to ''E. coli'' cells. Perhaps codon optimization and a better expression system are needed to make our PprI BioBrick functional.<br />
<br />
Finally, we have shown that PprA, when expressed in sufficient quantities, does appear to confer tolerance to ''E. coli''.<br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T21:07:05Z<p>Shao: /* Discussion */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
=== Damage Tolerance ===<br />
To measure the DNA damage tolerance conferred by each part, we used UV irradiation 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&deg;C. For detailed protocols, refer to the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].<br />
<br />
The tolerance parts tested were as follows:<br />
=====Parts containing one gene each=====<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
*CDS: PprI, PprA, PprM or RecA<br />
<br />
=====Parts containing two genes=====<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
*CDS1+2: PprI+RecA, PprA+RecA, PprM+RecA, PprI+PprA, PprI+PprM, PprA+PprM<br />
<br />
Previously we tested some of these parts without IPTG induction. With the rationale that IPTG induction may increase expression of the protective proteins, we repeated past characterization tests with the inclusion of IPTG addition, and obtained slightly different results. These are indicated in the chart below.<br />
<br />
[[File:2011_osaka_tolerance_results.png|800px]]<br />
<br />
==== Discussion ====<br />
===== Single-gene parts =====<br />
*As before, PprI did not appear to significantly increase tolerance, corroborating with its known role as an inducer of other radiotolerance proteins. As ''E. coli'' would lack these required ''D. radiodurans'' proteins it is expected that PprI would not be able to confer tolerance on its own.<br />
*On the other hand, PprA, once induced with IPTG, seemed to confer a degree of DNA damage tolerance to ''E. coli''. This is not surprising considering PprA's function as a direct executor of DNA damage repair.<br />
*''D. radiodurans'' RecA was indicated in our previous experimental results to confer the highest tolerance among the four radiotolerance genes; however, here, its effect was less than that of either PprA or PprM. This might be because it plays a complementary role to native ''E. coli'' RecA, thus the higher expression level induced by IPTG was of no additional benefit.<br />
*Perhaps the most unexpected result was that from PprM. In our previous experiment (using non-induced parts), PprM also appeared to confer a degree of tolerance although it was not significant enough to be of note. Here, IPTG-induced PprM levels seem to provide a significant amount of DNA damage tolerance. This is somewhat in contradiction to literature which describes PprM as a modulator of the PprI-dependent damage response that depends on downstream effector proteins (PprA etc) to carry out its protective role.<br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T21:06:21Z<p>Shao: /* Damage Tolerance */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
=== Damage Tolerance ===<br />
To measure the DNA damage tolerance conferred by each part, we used UV irradiation 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&deg;C. For detailed protocols, refer to the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].<br />
<br />
The tolerance parts tested were as follows:<br />
=====Parts containing one gene each=====<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
*CDS: PprI, PprA, PprM or RecA<br />
<br />
=====Parts containing two genes=====<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
*CDS1+2: PprI+RecA, PprA+RecA, PprM+RecA, PprI+PprA, PprI+PprM, PprA+PprM<br />
<br />
Previously we tested some of these parts without IPTG induction. With the rationale that IPTG induction may increase expression of the protective proteins, we repeated past characterization tests with the inclusion of IPTG addition, and obtained slightly different results. These are indicated in the chart below.<br />
<br />
[[File:2011_osaka_tolerance_results.png|800px]]<br />
<br />
==== Discussion ====<br />
As before, PprI did not appear to significantly increase tolerance, corroborating with its known role as an inducer of other radiotolerance proteins. As ''E. coli'' would lack these required ''D. radiodurans'' proteins it is expected that PprI would not be able to confer tolerance on its own.<br />
On the other hand, PprA, once induced with IPTG, seemed to confer a degree of DNA damage tolerance to ''E. coli''. This is not surprising considering PprA's function as a direct executor of DNA damage repair.<br />
''D. radiodurans'' RecA was indicated in our previous experimental results to confer the highest tolerance among the four radiotolerance genes; however, here, its effect was less than that of either PprA or PprM. This might be because it plays a complementary role to native ''E. coli'' RecA, thus the higher expression level induced by IPTG was of no additional benefit.<br />
Perhaps the most unexpected result was that from PprM. In our previous experiment (using non-induced parts), PprM also appeared to confer a degree of tolerance although it was not significant enough to be of note. Here, IPTG-induced PprM levels seem to provide a significant amount of DNA damage tolerance. This is somewhat in contradiction to literature which describes PprM as a modulator of the PprI-dependent damage response that depends on downstream effector proteins (PprA etc) to carry out its protective role.<br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T20:53:15Z<p>Shao: /* Damage Tolerance */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
=== Damage Tolerance ===<br />
To measure the DNA damage tolerance conferred by each part, we used UV irradiation 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&deg;C. For detailed protocols, refer to the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].<br />
<br />
The tolerance parts tested were as follows:<br />
=====Parts containing one gene each=====<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
*CDS: PprI, PprA, PprM or RecA<br />
<br />
=====Parts containing two genes=====<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
*CDS1+2: PprI+RecA, PprA+RecA, PprM+RecA, PprI+PprA, PprI+PprM, PprA+PprM<br />
<br />
Previously we tested some of these parts without IPTG induction. With the rationale that IPTG induction may increase expression of the protective proteins, we repeated past characterization tests with the inclusion of IPTG addition, and obtained slightly different results. These are indicated in the charts below.<br />
<br />
[[File:2011_osaka_tolerance_results.png|800px]]<br />
<br />
The PprA protein mainly functions in repair of blunt-ended DNA double-strand breaks. Since UV exposure mainly causes thymine dimerization but not strand breaks, it may appear that PprA does not protect against UV but may confer tolerance against other forms of DNA damage such as that induced by ionizing radiation or Mitomycin C.<br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/File:2011_osaka_tolerance_results.pngFile:2011 osaka tolerance results.png2011-10-28T20:52:15Z<p>Shao: </p>
<hr />
<div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T20:16:00Z<p>Shao: /* Parts containing two genes */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
=== Damage Tolerance ===<br />
To measure the DNA damage tolerance conferred by each part, we used UV irradiation 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&deg;C. For detailed protocols, refer to the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].<br />
<br />
The tolerance parts tested were as follows:<br />
=====Parts containing one gene each=====<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
*CDS: PprI, PprA, PprM or RecA<br />
<br />
=====Parts containing two genes=====<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
*CDS1+2: PprI+RecA, PprA+RecA, PprM+RecA, PprI+PprA, PprI+PprM, PprA+PprM<br />
<br />
Previously we tested some of these parts without IPTG induction. With the rationale that IPTG induction may increase expression of the protective proteins, we repeated past characterization tests with the inclusion of IPTG addition, and obtained slightly different results. These are indicated in the charts below.<br />
<br />
(charts)<br />
<br />
The PprA protein mainly functions in repair of blunt-ended DNA double-strand breaks. Since UV exposure mainly causes thymine dimerization but not strand breaks, it may appear that PprA does not protect against UV but may confer tolerance against other forms of DNA damage such as that induced by ionizing radiation or Mitomycin C.<br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T20:15:43Z<p>Shao: /* Parts containing one gene each */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
=== Damage Tolerance ===<br />
To measure the DNA damage tolerance conferred by each part, we used UV irradiation 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&deg;C. For detailed protocols, refer to the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].<br />
<br />
The tolerance parts tested were as follows:<br />
=====Parts containing one gene each=====<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
*CDS: PprI, PprA, PprM or RecA<br />
<br />
=====Parts containing two genes=====<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
**CDS1+2: PprI+RecA, PprA+RecA, PprM+RecA, PprI+PprA, PprI+PprM, PprA+PprM<br />
<br />
Previously we tested some of these parts without IPTG induction. With the rationale that IPTG induction may increase expression of the protective proteins, we repeated past characterization tests with the inclusion of IPTG addition, and obtained slightly different results. These are indicated in the charts below.<br />
<br />
(charts)<br />
<br />
The PprA protein mainly functions in repair of blunt-ended DNA double-strand breaks. Since UV exposure mainly causes thymine dimerization but not strand breaks, it may appear that PprA does not protect against UV but may confer tolerance against other forms of DNA damage such as that induced by ionizing radiation or Mitomycin C.<br />
<br />
<br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T20:15:19Z<p>Shao: /* Tests */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
=== Damage Tolerance ===<br />
To measure the DNA damage tolerance conferred by each part, we used UV irradiation 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&deg;C. For detailed protocols, refer to the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].<br />
<br />
The tolerance parts tested were as follows:<br />
=====Parts containing one gene each=====<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
**CDS: PprI, PprA, PprM or RecA<br />
<br />
=====Parts containing two genes=====<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
**CDS1+2: PprI+RecA, PprA+RecA, PprM+RecA, PprI+PprA, PprI+PprM, PprA+PprM<br />
<br />
Previously we tested some of these parts without IPTG induction. With the rationale that IPTG induction may increase expression of the protective proteins, we repeated past characterization tests with the inclusion of IPTG addition, and obtained slightly different results. These are indicated in the charts below.<br />
<br />
(charts)<br />
<br />
The PprA protein mainly functions in repair of blunt-ended DNA double-strand breaks. Since UV exposure mainly causes thymine dimerization but not strand breaks, it may appear that PprA does not protect against UV but may confer tolerance against other forms of DNA damage such as that induced by ionizing radiation or Mitomycin C.<br />
<br />
<br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T20:14:25Z<p>Shao: /* Tests */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
=== Damage Tolerance ===<br />
To measure the DNA damage tolerance conferred by each part, we used UV irradiation 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&deg;C. For detailed protocols, refer to the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].<br />
<br />
The tolerance parts tested were as follows:<br />
*Parts containing one gene each<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
**CDS: PprI, PprA, PprM or RecA<br />
<br />
*Parts containing two genes<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
**CDS1+2: PprI+RecA, PprA+RecA, PprM+RecA, PprI+PprA, PprI+PprM, PprA+PprM<br />
<br />
Previously we tested some of these parts without IPTG induction. With the rationale that IPTG induction may increase expression of the protective proteins, we repeated past characterization tests with the inclusion of IPTG addition, and obtained slightly different results. These are indicated in the charts below.<br />
<br />
(charts)<br />
<br />
The PprA protein mainly functions in repair of blunt-ended DNA double-strand breaks. Since UV exposure mainly causes thymine dimerization but not strand breaks, it may appear that PprA does not protect against UV but may confer tolerance against other forms of DNA damage such as that induced by ionizing radiation or Mitomycin C.<br />
<br />
<br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T20:06:33Z<p>Shao: /* Cell viability */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
=== Damage Tolerance ===<br />
To measure the DNA damage tolerance conferred by each part, we used UV irradiation 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&deg;C. For detailed protocols, refer to the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:58:54Z<p>Shao: /* Project Details */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
<!--<br />
ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
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</html><br />
<div class="padding"><br />
== Project Details==<br />
=== DNA damage tolerance===<br />
==== <i>D. radiodurans</i> ====<br />
[[File:deinococcus.jpg|200px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of DNA damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. It is an aerobically-growing bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, <i>E. coli</i> can withstand up to 200 Gy, and an acute exposure of just 5-10 Gy is lethal to a human being.</p><br />
<p>We explored various genes from <i>D. radiodurans</i>, implicated in its remarkable DNA damage resistance. By BioBricking selected genes and transforming them into <i>E. coli</i>, we hoped to confer additional DNA damage tolerance to the host cells.</p><br />
<br />
<br />
<br />
==== Radiotolerance genes ====<br />
*'''PprI'''<br />
PprI, which is unique to ''D. radiodurans'', is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.<br />
<br />
*'''PprA'''<br />
A pleiotropic protein promoting DNA repair, its role in radiation resistance of ''Deinococcus radiodurans''<br />
was demonstrated.<br />
PprA preferentially binds to double-stranded DNA carrying strand breaks, inhibits ''E. coli'' exonuclease III activity, and stimulates the DNA end-joining reaction catalysed by ATP-dependent and NAD-dependent DNA ligases. These<br />
results suggest that ''D. radiodurans'' has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.<br />
<br />
*'''PprM'''<br />
PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp). PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from ''D. geothermalis'' and ''Thermus thermophilus''. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in ''D. radiodurans''.<br />
<br />
*'''RecA'''<br />
The ''D. radiodurans'' RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the ''E. coli'' RecA protein. ''D. radiodurans'' recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that ''E. coli'' RecA did not complement an IR-sensitive ''D. radiodurans'' recA point-mutant (rec30) and that expression of ''D. radiodurans'' RecA in ''E. coli'' was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a ''D. radiodurans'' recA null mutant (Schlesinger, 2007).<br />
<br />
<br />
<br />
<br />
=== DNA damage detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.</p><br />
<br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>Our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:58:13Z<p>Shao: /* Radiotolerance genes */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
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ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
dd {margin:0 0 0 0;}<br />
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</style><br />
</html><br />
<div class="padding"><br />
== Project Details==<br />
=== DNA damage tolerance===<br />
==== <i>D. radiodurans</i> ====<br />
[[File:deinococcus.jpg|200px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of DNA damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. It is an aerobically-growing bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, <i>E. coli</i> can withstand up to 200 Gy, and an acute exposure of just 5-10 Gy is lethal to a human being.</p><br />
<p>We explored various genes from <i>D. radiodurans</i>, implicated in its remarkable DNA damage resistance. By BioBricking selected genes and transforming them into <i>E. coli</i>, we hoped to confer additional DNA damage tolerance to the host cells.</p><br />
<br />
<br />
<br />
==== Radiotolerance genes ====<br />
*'''PprI'''<br />
PprI, which is unique to ''D. radiodurans'', is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.<br />
<br />
*'''PprA'''<br />
A pleiotropic protein promoting DNA repair, its role in radiation resistance of ''Deinococcus radiodurans''<br />
was demonstrated.<br />
PprA preferentially binds to double-stranded DNA carrying strand breaks, inhibits ''E. coli'' exonuclease III activity, and stimulates the DNA end-joining reaction catalysed by ATP-dependent and NAD-dependent DNA ligases. These<br />
results suggest that ''D. radiodurans'' has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.<br />
<br />
*'''PprM'''<br />
PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp). PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from ''D. geothermalis'' and ''Thermus thermophilus''. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in ''D. radiodurans''.<br />
<br />
*'''RecA'''<br />
The ''D. radiodurans'' RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the ''E. coli'' RecA protein. ''D. radiodurans'' recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that ''E. coli'' RecA did not complement an IR-sensitive ''D. radiodurans'' recA point-mutant (rec30) and that expression of ''D. radiodurans'' RecA in ''E. coli'' was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a ''D. radiodurans'' recA null mutant (Schlesinger, 2007).<br />
<br />
=== DNA damage detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.</p><br />
<br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>Our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:57:28Z<p>Shao: /* Radiotolerance genes */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
<!--<br />
ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
dd {margin:0 0 0 0;}<br />
--><br />
</style><br />
</html><br />
<div class="padding"><br />
== Project Details==<br />
=== DNA damage tolerance===<br />
==== <i>D. radiodurans</i> ====<br />
[[File:deinococcus.jpg|200px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of DNA damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. It is an aerobically-growing bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, <i>E. coli</i> can withstand up to 200 Gy, and an acute exposure of just 5-10 Gy is lethal to a human being.</p><br />
<p>We explored various genes from <i>D. radiodurans</i>, implicated in its remarkable DNA damage resistance. By BioBricking selected genes and transforming them into <i>E. coli</i>, we hoped to confer additional DNA damage tolerance to the host cells.</p><br />
<br />
<br />
<br />
==== Radiotolerance genes ====<br />
*PprI<br />
PprI, which is unique to ''D. radiodurans'', is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.<br />
<br />
*PprA<br />
A pleiotropic protein promoting DNA repair, its role in radiation resistance of ''Deinococcus radiodurans''<br />
was demonstrated.<br />
PprA preferentially binds to double-stranded DNA carrying strand breaks, inhibits ''E. coli'' exonuclease III activity, and stimulates the DNA end-joining reaction catalysed by ATP-dependent and NAD-dependent DNA ligases. These<br />
results suggest that ''D. radiodurans'' has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.<br />
<br />
*PprM<br />
PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp). PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from ''D. geothermalis'' and ''Thermus thermophilus''. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in ''D. radiodurans''.<br />
<br />
*RecA<br />
The ''D. radiodurans'' RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the ''E. coli'' RecA protein. ''D. radiodurans'' recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that ''E. coli'' RecA did not complement an IR-sensitive ''D. radiodurans'' recA point-mutant (rec30) and that expression of ''D. radiodurans'' RecA in ''E. coli'' was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a ''D. radiodurans'' recA null mutant (Schlesinger, 2007).<br />
<br />
=== DNA damage detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.</p><br />
<br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>Our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:51:33Z<p>Shao: /* DNA damage tolerance */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
<!--<br />
ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
dd {margin:0 0 0 0;}<br />
--><br />
</style><br />
</html><br />
<div class="padding"><br />
== Project Details==<br />
=== DNA damage tolerance===<br />
==== <i>D. radiodurans</i> ====<br />
[[File:deinococcus.jpg|200px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of DNA damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. It is an aerobically-growing bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, <i>E. coli</i> can withstand up to 200 Gy, and an acute exposure of just 5-10 Gy is lethal to a human being.</p><br />
<p>We explored various genes from <i>D. radiodurans</i>, implicated in its remarkable DNA damage resistance. By BioBricking selected genes and transforming them into <i>E. coli</i>, we hoped to confer additional DNA damage tolerance to the host cells.</p><br />
<br />
<br />
<br />
==== Radiotolerance genes ====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== DNA damage detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.</p><br />
<br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>Our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:50:42Z<p>Shao: /* Project Details */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
<!--<br />
ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
dd {margin:0 0 0 0;}<br />
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</style><br />
</html><br />
<div class="padding"><br />
== Project Details==<br />
=== DNA damage tolerance===<br />
[[File:deinococcus.jpg|200px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of DNA damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. It is an aerobically-growing bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, <i>E. coli</i> can withstand up to 200 Gy, and an acute exposure of just 5-10 Gy is lethal to a human being.</p><br />
<p>We explored various genes from <i>D. radiodurans</i>, implicated in its remarkable DNA damage resistance. By BioBricking selected genes and transforming them into <i>E. coli</i>, we hoped to confer additional DNA damage tolerance to the host cells.</p><br />
<br />
<br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== DNA damage detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.</p><br />
<br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>Our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:50:20Z<p>Shao: /* DNA damage tolerance */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
<!--<br />
ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
dd {margin:0 0 0 0;}<br />
--><br />
</style><br />
</html><br />
<div class="padding"><br />
== Project Details==<br />
=== DNA damage tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of DNA damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens. It is an aerobically-growing bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, <i>E. coli</i> can withstand up to 200 Gy, and an acute exposure of just 5-10 Gy is lethal to a human being.</p><br />
<p>We explored various genes from <i>D. radiodurans</i>, implicated in its remarkable DNA damage resistance. By BioBricking selected genes and transforming them into <i>E. coli</i>, we hoped to confer additional DNA damage tolerance to the host cells.</p><br />
<br />
<br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== DNA damage detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.</p><br />
<br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>Our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:46:49Z<p>Shao: /* DNA Damage Tolerance */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
<!--<br />
ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
dd {margin:0 0 0 0;}<br />
--><br />
</style><br />
</html><br />
<div class="padding"><br />
== Project Details==<br />
=== DNA damage tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.It is an aerobic bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, an acute exposure of just 5-10 Gy is lethal to the average human. </p><br />
<p>We explored various genes from <i>D. radiodurans</i>, implicated in its remarkable DNA damage resistance. By BioBricking selected genes and transforming them into <i>E. coli</i>, we hoped to confer additional DNA damage tolerance to the host cells.</p><br />
<br />
<br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== DNA damage detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.</p><br />
<br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>Our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:44:37Z<p>Shao: /* DNA Damage Detection */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
<!--<br />
ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
dd {margin:0 0 0 0;}<br />
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</style><br />
</html><br />
<div class="padding"><br />
== Project Details==<br />
=== DNA Damage Tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.It is an aerobic bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, an acute exposure of just 5-10 Gy is lethal to the average human. </p><br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== DNA damage detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.</p><br />
<br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>Our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:44:12Z<p>Shao: /* DNA Damage Detection */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
<!--<br />
ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
dd {margin:0 0 0 0;}<br />
--><br />
</style><br />
</html><br />
<div class="padding"><br />
== Project Details==<br />
=== DNA Damage Tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.It is an aerobic bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, an acute exposure of just 5-10 Gy is lethal to the average human. </p><br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== DNA Damage Detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.</p><br />
<br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>Our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:43:55Z<p>Shao: /* DNA Damage Detection */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
<!--<br />
ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
dd {margin:0 0 0 0;}<br />
--><br />
</style><br />
</html><br />
<div class="padding"><br />
== Project Details==<br />
=== DNA Damage Tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.It is an aerobic bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, an acute exposure of just 5-10 Gy is lethal to the average human. </p><br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== DNA Damage Detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.</p><br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>Our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:42:18Z<p>Shao: /* Project Details */</p>
<hr />
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<div class="padding"><br />
== Project Details==<br />
=== DNA Damage Tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.It is an aerobic bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, an acute exposure of just 5-10 Gy is lethal to the average human. </p><br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== DNA Damage Detection ===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.<br />
In addition, our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). Therefore we decided to employ lycopene biosynthesis as a reporter.</p><br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:41:31Z<p>Shao: /* SOS response */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
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</html><br />
<div class="padding"><br />
== Project Details==<br />
=== 1. Damage tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.It is an aerobic bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, an acute exposure of just 5-10 Gy is lethal to the average human. </p><br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== 2. Detection of DNA damage===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the promoter of the RecA gene ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. While RecA is an inducer of SOS genes, it itself is an SOS gene that is auto-induced upon DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.<br />
In addition, our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). Therefore we decided to employ lycopene biosynthesis as a reporter.</p><br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:40:16Z<p>Shao: /* Lycopene biosynthesis */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
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dd {margin:0 0 0 0;}<br />
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</style><br />
</html><br />
<div class="padding"><br />
== Project Details==<br />
=== 1. Damage tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.It is an aerobic bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, an acute exposure of just 5-10 Gy is lethal to the average human. </p><br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== 2. Detection of DNA damage===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the RecA promoter([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.<br />
In addition, our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). Therefore we decided to employ lycopene biosynthesis as a reporter.</p><br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by 2009 Cambridge (<partinfo>K274100</partinfo>).<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:39:52Z<p>Shao: /* Lycopene biosynthesis */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
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</html><br />
<div class="padding"><br />
== Project Details==<br />
=== 1. Damage tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.It is an aerobic bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, an acute exposure of just 5-10 Gy is lethal to the average human. </p><br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== 2. Detection of DNA damage===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the RecA promoter([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.<br />
In addition, our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). Therefore we decided to employ lycopene biosynthesis as a reporter.</p><br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<p>As shown in the figure on the right, the heterologous enzymes CrtE, CrtB, CrtI were introduced into <i>E. coli</i> to complete the biosynthesis of lycopene. We used a lycopene biosynthetic gene cluster provided by Cambridge (<partinfo>K274100</partinfo>)<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:33:44Z<p>Shao: /* Lycopene biosynthesis */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
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ul {list-style:disc;}<br />
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dd {margin:0 0 0 0;}<br />
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</html><br />
<div class="padding"><br />
== Project Details==<br />
=== 1. Damage tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.It is an aerobic bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, an acute exposure of just 5-10 Gy is lethal to the average human. </p><br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== 2. Detection of DNA damage===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the RecA promoter([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.<br />
In addition, our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). Therefore we decided to employ lycopene biosynthesis as a reporter.</p><br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.</p><br />
<p>Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP), which can be biologically produced by two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/ProjectTeam:Osaka/Project2011-10-28T19:32:46Z<p>Shao: /* 2. Detection of DNA damage */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<html><br />
<style type="text/css"><br />
<!--<br />
ul {list-style:disc;}<br />
dt {font-weight:bold;}<br />
dd {margin:0 0 0 0;}<br />
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</html><br />
<div class="padding"><br />
== Project Details==<br />
=== 1. Damage tolerance===<br />
[[File:deinococcus.jpg|250px|right|''D.radiodurans'']]<br />
<p><br />
The bacterium ''Deinococcus radiodurans'' shows remarkable resistance to a range of damage caused by ionizing radiation, desiccation, UV radiation, oxidizing agents, and electrophilic mutagens.It is an aerobic bacterium that is most famous for its extreme resistance to ionizing radiation; it not only can survive acute exposures to gamma radiation that exceed 15,000 Gy, but it can also grow continuously in the presence of chronic radiation (60 Gy/hour) without any effect on its growth rate or ability to express cloned genes. For comparison, an acute exposure of just 5-10 Gy is lethal to the average human. </p><br />
<br />
====Factors====<br />
<html><br />
<ul><br />
<li><br />
<dl><br />
<dt>recA</dt><br />
<dd>The <I>D. radiodurans</I> RecA protein has been characterized and its gene has been sequenced; it shows greater than 50% identity to the <I>E. coli</I> RecA protein. <I>D. radiodurans</I> recA mutants are highly sensitive to UV and ionizing radiation. In this context, early work by Carroll et al (1996) reported that <I>E. coli</I> RecA did not complement an IR-sensitive <I>D. radiodurans</I> recA point-mutant (rec30) and that expression of <I>D. radiodurans</I> RecA in <I>E. coli</I> was lethal. More recently, however, it has been reported that <I>E. coli</I> recA can provide partial complementation to a <I>D. radiodurans</I> recA null mutant (Schlesinger, 2007).</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprI</dt><br />
<dd>PprI, which is unique to <I>D. radiodurans</I>, is invoked by present data as the most important protein for radiation response mechanism.<br />
PprI can significantly and specifically induce the gene expression of recA and pprA and enhance the enzyme<br />
activities of catalases. These results strongly suggest that PprI plays a crucial role in regulating multiple DNA repair and protection<br />
pathways in response to radiation stress.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprA</dt><br />
<dd>A pleiotropic protein promoting DNA repair, role in radiation resistance of <I>Deinococcus radiodurans</I><br />
was demonstrated.<br />
PprA preferentially bound to double-stranded DNA carrying strand breaks, inhibited <I>E. coli</I> exonuclease III activity, and stimulated the DNA end-joining reaction catalysed by ATPdependent and NAD-dependent DNA ligases. These<br />
results suggest that <I>D. radiodurans</I> has a radiationinduced non-homologous end-joining repair mechanism<br />
in which PprA plays a critical role.</dd><br />
</dl></li><br />
<li><br />
<dl><br />
<dt>pprM</dt><br />
<dd>PprM (a modulator of the PprI-dependent DNA damage response) is a homolog of cold shock protein (Csp)PprM regulates the induction of PprA but not that of RecA. PprM belongs in a distinct clade of a subfamily together with Csp homologs from <I>D. geothermalis</I> and Thermus thermophilus. <br />
PprM plays an important role in the induction of RecA and PprA and is involved in the unique radiation response mechanism controlled by PprI in <I>D. radiodurans</I>.</dd><br />
</dl></li><br />
</ul><br />
</html><br />
<br />
=== 2. Detection of DNA damage===<br />
==== SOS response ====<br />
[[File:SOS response.png|left|sos responce|300px]]<br />
<p>If DNA is significantly damaged (eg by exposure to UV radiation or chemicals), synthesis of several DNA damage-related proteins occurs quickly.<br />
This reaction to DNA damage is the SOS response.</p><br />
<p>RecA is a 38 kilodalton <I>Escherichia coli</I> protein essential for the repair and maintenance of DNA. RecA has multiple activities, all related to DNA repair. In the bacterial SOS response, it has a co-protease function in the autocatalytic cleavage of the LexA repressor and the λ repressor. LexA is expressed constitutively and prevents expression of damage-related proteins by binding to SOS box as a repressor. RecA is activated by binding to single-stranded DNA, and the activated RecA then turns on LexA protease activity. Self-cleavage of LexA derepresses the expression of damage-related proteins enabling a response to be mounted. </p><br />
<p>We decided to employ the RecA promoter([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 BBa J22106]) to detect DNA damage. Expression of genes downstream of this promoter is induced by DNA damage.<br />
In addition, our Bio-dosimeter must have some sort of visible output to alert users to radioactivity (detected as DNA damage). Therefore we decided to employ lycopene biosynthesis as a reporter.</p><br />
<br />
==== Lycopene biosynthesis ====<br />
[[File:Pigment.jpg|280px|right]]<br />
<p>In a previous iGEM project, "colrcoli", we attempted to use <I>E.coli</I> as a paint tool. To that end, we examined biosynthesis of carotenoid pigments as a way of producing color. Here, we attempted to use biosynthesis of the carotenoid lycopene as a reporter for DNA damage.</p><br />
<p>Carotenoid is a family of natural pigments. Many plants such as fruits and vegetables contain these pigments. For example, tomato has lycopene(red), carrot has carotene(orange). Xanthophyll(yellow) is found in almost all plants.<br />
<br />
Biosynthesis of carotenoid pigments starts from FPP(FARNESYL DIPHOSPHATE). FPP is formed from isopentenylpyrophosphate(IPP) and dimethylallylpyrophosphate(DMAPP). IPP and DMAPP are formed through two distinct pathways, the mevalonate and non-mevalonate pathways. In <i>E.coli</i>, FPP is formed through the non-mevalonate pathway. By the introduction of heterologous enzymatic genes colorless FPP is then converted to orange-red lycopene, which has a peak absorbance at 407nm that is easily measured. </p><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br><br />
<br />
=== References===<br />
<ol><br />
<li>[1] Y. Hua et al., “PprI: a general switch responsible for extreme radioresistance of ''Deinococcus radiodurans'',” Biochemical and Biophysical Research Communications, vol. 306, no. 2, pp. 354-360, Jun. 2003.</li><br />
<li>G. Gao, B. Tian, L. Liu, D. Sheng, B. Shen, and Y. Hua, “Expression of ''Deinococcus radiodurans'' PprI enhances the radioresistance of ''Escherichia coli'',” DNA Repair, vol. 2, no. 12, pp. 1419-1427, Dec. 2003.</li><br />
<li>I. Narumi, K. Satoh, S. Cui, T. Funayama, S. Kitayama, and H. Watanabe, “PprA: a novel protein from ''Deinococcus radiodurans'' that stimulates DNA ligation,” Molecular Microbiology, vol. 54, no. 1, pp. 278-285, Oct. 2004.</li><br />
<li>S. Kota and H. S. Misra, “PprA: A protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'',” Applied Microbiology and Biotechnology, vol. 72, no. 4, pp. 790-796, Oct. 2006.</li><br />
<li>H. Lu et al., “''Deinococcus radiodurans'' PprI switches on DNA damage response and cellular survival networks after radiation damage,” Molecular & Cellular Proteomics: MCP, vol. 8, no. 3, pp. 481-494, Mar. 2009.</li><br />
<li>H. Ohba, K. Satoh, H. Sghaier, T. Yanagisawa, and I. Narumi, “Identification of PprM: a modulator of the PprI-dependent DNA damage response in ''Deinococcus radiodurans'',” Extremophiles: Life Under Extreme Conditions, vol. 13, no. 3, pp. 471-479, May 2009.</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:25:35Z<p>Shao: /* Tests */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:24:12Z<p>Shao: /* Cell viability */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== DNA damage tolerance ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.<br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:23:41Z<p>Shao: /* Cell viability */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== Cell viability ====<br />
*We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!<br />
*To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!<br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.<br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:23:15Z<p>Shao: /* Damage detection */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== Cell viability ====<br />
<p>We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!</p><br />
<p>To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!</p><br />
<br />
==== Damage detection ====<br />
* We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.<br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:22:03Z<p>Shao: /* Cell viability */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== Cell viability ====<br />
<p>We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!</p><br />
<p>To more properly measure the tolerance conferred by each part against DNA ''double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!</p><br />
<br />
==== SOS response ====<br />
<p>We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:21:30Z<p>Shao: /* Future work */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Work in Progress ===<br />
==== Cell viability ====<br />
<p>We are working on assembling a device with all four tolerance genes (PprI, PprA, PprM, RecA) but will not have time to characterize it properly before the wiki freeze. Stay tuned for our poster/presentation at the iGEM World Championship Jamboree for the results!</p><br />
<p>To more properly measure the tolerance conferred by each part against ''DNA double strand breaks'' (the primary effect of ionizing radiation), we are working on characterizing the parts' tolerances against the drug Mitomycin C. Again, results will be too late for the wiki freeze so catch our presentation at the World Championship Jamboree!</p><br />
<br />
==== SOS response ====<br />
<p>We have assembled a device utilizing GFP as reporter, but did not have time to characterize it properly. The results will be in our final presentation.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:13:38Z<p>Shao: /* SOS promoter assay */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. For details check the [https://2011.igem.org/Team:Osaka/Protocols Protocols page].</p><br />
<p>[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]</p><br />
<br />
<p>Response was defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.<br />
We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Future work ===<br />
<p>'''Cell viability'''</p><br />
<p>We created some parts (PprI , PprA , PprM , RecA) but did not have enough time to evaluate them completely.</p><br />
<p>The effects of each parts to cell viability are shown above, but we will be able to evaluate them more precisely by measuring the effects of other combined parts. (We have already assayed a combined part, mix(all four parts combined).)</p><br />
<p>We should test responses of cells to other damage types, too.</p><br />
<p>'''SOS response'''</p><br />
<p>Our project is "Bio-dosimeter", so we should construct two devices about damage tolerance and damage detection respectively.</p><br />
<p>Our constructed bio-dosimeter shows the level of radiation by one pigment, but it's somewhat difficult to identify the level from light and shade of one color.Therefore, in future, Bio-dosimeter should be going to show the level of radiation using several pigments and promoters.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:11:44Z<p>Shao: /* SOS promoter assay */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.</p><br />
[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]<br />
<br />
<p>Response is defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.</p><br />
<p>We observed a response to UV irradiation that increased with energy dosage from 200 to 600 J/m^2. Response was decreased at 800 J/m^2, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Future work ===<br />
<p>'''Cell viability'''</p><br />
<p>We created some parts (PprI , PprA , PprM , RecA) but did not have enough time to evaluate them completely.</p><br />
<p>The effects of each parts to cell viability are shown above, but we will be able to evaluate them more precisely by measuring the effects of other combined parts. (We have already assayed a combined part, mix(all four parts combined).)</p><br />
<p>We should test responses of cells to other damage types, too.</p><br />
<p>'''SOS response'''</p><br />
<p>Our project is "Bio-dosimeter", so we should construct two devices about damage tolerance and damage detection respectively.</p><br />
<p>Our constructed bio-dosimeter shows the level of radiation by one pigment, but it's somewhat difficult to identify the level from light and shade of one color.Therefore, in future, Bio-dosimeter should be going to show the level of radiation using several pigments and promoters.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:10:59Z<p>Shao: /* SOS promoter assay */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]), by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light and then incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.</p><br />
[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]<br />
<br />
<p>Response is defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.</p><br />
<p>We observed a response to UV irradiation that increased with energy dosage from 200 to 400 J/m^2. Response decreased with higher energy dosages, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Future work ===<br />
<p>'''Cell viability'''</p><br />
<p>We created some parts (PprI , PprA , PprM , RecA) but did not have enough time to evaluate them completely.</p><br />
<p>The effects of each parts to cell viability are shown above, but we will be able to evaluate them more precisely by measuring the effects of other combined parts. (We have already assayed a combined part, mix(all four parts combined).)</p><br />
<p>We should test responses of cells to other damage types, too.</p><br />
<p>'''SOS response'''</p><br />
<p>Our project is "Bio-dosimeter", so we should construct two devices about damage tolerance and damage detection respectively.</p><br />
<p>Our constructed bio-dosimeter shows the level of radiation by one pigment, but it's somewhat difficult to identify the level from light and shade of one color.Therefore, in future, Bio-dosimeter should be going to show the level of radiation using several pigments and promoters.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:09:41Z<p>Shao: /* SOS promoter assay */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the RecA promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]) by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light, and then, incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.</p><br />
[[File:2011_osaka_promoter_1.png|400px]]<br />
[[File:2011_osaka_promoter_2.png|400px]]<br />
<br />
<p>Response is defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.</p><br />
<p>We observed a response to UV irradiation that increased with energy dosage from 200 to 400 J/m^2. Response decreased with higher energy dosages, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Future work ===<br />
<p>'''Cell viability'''</p><br />
<p>We created some parts (PprI , PprA , PprM , RecA) but did not have enough time to evaluate them completely.</p><br />
<p>The effects of each parts to cell viability are shown above, but we will be able to evaluate them more precisely by measuring the effects of other combined parts. (We have already assayed a combined part, mix(all four parts combined).)</p><br />
<p>We should test responses of cells to other damage types, too.</p><br />
<p>'''SOS response'''</p><br />
<p>Our project is "Bio-dosimeter", so we should construct two devices about damage tolerance and damage detection respectively.</p><br />
<p>Our constructed bio-dosimeter shows the level of radiation by one pigment, but it's somewhat difficult to identify the level from light and shade of one color.Therefore, in future, Bio-dosimeter should be going to show the level of radiation using several pigments and promoters.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:08:29Z<p>Shao: /* SOS promoter assay */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the RecA promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]) by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light, and then, incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.</p><br />
[[File:2011_osaka_promoter_1.png]]<br />
[[File:2011_osaka_promoter_2.png]]<br />
<br />
<p>Response is defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.</p><br />
<p>We observed a response to UV irradiation that increased with energy dosage from 200 to 400 J/m^2. Response decreased with higher energy dosages, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Future work ===<br />
<p>'''Cell viability'''</p><br />
<p>We created some parts (PprI , PprA , PprM , RecA) but did not have enough time to evaluate them completely.</p><br />
<p>The effects of each parts to cell viability are shown above, but we will be able to evaluate them more precisely by measuring the effects of other combined parts. (We have already assayed a combined part, mix(all four parts combined).)</p><br />
<p>We should test responses of cells to other damage types, too.</p><br />
<p>'''SOS response'''</p><br />
<p>Our project is "Bio-dosimeter", so we should construct two devices about damage tolerance and damage detection respectively.</p><br />
<p>Our constructed bio-dosimeter shows the level of radiation by one pigment, but it's somewhat difficult to identify the level from light and shade of one color.Therefore, in future, Bio-dosimeter should be going to show the level of radiation using several pigments and promoters.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/File:2011_osaka_promoter_1.pngFile:2011 osaka promoter 1.png2011-10-28T19:07:36Z<p>Shao: </p>
<hr />
<div></div>Shaohttp://2011.igem.org/File:2011_osaka_promoter_2.pngFile:2011 osaka promoter 2.png2011-10-28T19:07:27Z<p>Shao: </p>
<hr />
<div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:04:27Z<p>Shao: </p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|166px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|400px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the RecA promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]) by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light, and then, incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.</p><br />
[[File:SOS_promoter_response.png|450px|center]]<br />
<br />
<p>Response is defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.</p><br />
<p>We observed a response to UV irradiation that increased with energy dosage from 200 to 400 J/m^2. Response decreased with higher energy dosages, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Future work ===<br />
<p>'''Cell viability'''</p><br />
<p>We created some parts (PprI , PprA , PprM , RecA) but did not have enough time to evaluate them completely.</p><br />
<p>The effects of each parts to cell viability are shown above, but we will be able to evaluate them more precisely by measuring the effects of other combined parts. (We have already assayed a combined part, mix(all four parts combined).)</p><br />
<p>We should test responses of cells to other damage types, too.</p><br />
<p>'''SOS response'''</p><br />
<p>Our project is "Bio-dosimeter", so we should construct two devices about damage tolerance and damage detection respectively.</p><br />
<p>Our constructed bio-dosimeter shows the level of radiation by one pigment, but it's somewhat difficult to identify the level from light and shade of one color.Therefore, in future, Bio-dosimeter should be going to show the level of radiation using several pigments and promoters.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:01:54Z<p>Shao: /* Cell viability */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png|180px]]<br />
<br />
[[File:2011_osaka_tolerance_2.png|180px]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the RecA promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]) by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light, and then, incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.</p><br />
[[File:SOS_promoter_response.png|450px|center]]<br />
<br />
<p>Response is defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.</p><br />
<p>We observed a response to UV irradiation that increased with energy dosage from 200 to 400 J/m^2. Response decreased with higher energy dosages, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Future work ===<br />
<p>'''Cell viability'''</p><br />
<p>We created some parts (PprI , PprA , PprM , RecA) but did not have enough time to evaluate them completely.</p><br />
<p>The effects of each parts to cell viability are shown above, but we will be able to evaluate them more precisely by measuring the effects of other combined parts. (We have already assayed a combined part, mix(all four parts combined).)</p><br />
<p>We should test responses of cells to other damage types, too.</p><br />
<p>'''SOS response'''</p><br />
<p>Our project is "Bio-dosimeter", so we should construct two devices about damage tolerance and damage detection respectively.</p><br />
<p>Our constructed bio-dosimeter shows the level of radiation by one pigment, but it's somewhat difficult to identify the level from light and shade of one color.Therefore, in future, Bio-dosimeter should be going to show the level of radiation using several pigments and promoters.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T19:00:52Z<p>Shao: /* Cell viability */</p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.<br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png]]<br />
<br />
[[File:2011_osaka_tolerance_2.png]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
===SOS promoter assay===<br />
<p>We assayed the RecA promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]) by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light, and then, incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.</p><br />
[[File:SOS_promoter_response.png|450px|center]]<br />
<br />
<p>Response is defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.</p><br />
<p>We observed a response to UV irradiation that increased with energy dosage from 200 to 400 J/m^2. Response decreased with higher energy dosages, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Future work ===<br />
<p>'''Cell viability'''</p><br />
<p>We created some parts (PprI , PprA , PprM , RecA) but did not have enough time to evaluate them completely.</p><br />
<p>The effects of each parts to cell viability are shown above, but we will be able to evaluate them more precisely by measuring the effects of other combined parts. (We have already assayed a combined part, mix(all four parts combined).)</p><br />
<p>We should test responses of cells to other damage types, too.</p><br />
<p>'''SOS response'''</p><br />
<p>Our project is "Bio-dosimeter", so we should construct two devices about damage tolerance and damage detection respectively.</p><br />
<p>Our constructed bio-dosimeter shows the level of radiation by one pigment, but it's somewhat difficult to identify the level from light and shade of one color.Therefore, in future, Bio-dosimeter should be going to show the level of radiation using several pigments and promoters.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/Team:Osaka/TestsTeam:Osaka/Tests2011-10-28T18:59:51Z<p>Shao: </p>
<hr />
<div>{{Osaka}}<br />
__NOTOC__<br />
<div class="padding"><br />
== Tests ==<br />
===Cell viability===<br />
<p>We performed the UV assay. The cells were plated on respective 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&deg;C. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.</p><br />
<br />
<br />
[[File:2011_osaka_tolerance_1.png]]<br />
[[File:2011_osaka_tolerance_2.png]]<br />
<br />
<p>PprM gene could also confer high tolerance to inserted cells. </p><br />
<p>We expected that all cells inserted each genes could increase its ratio of cell survival, however, two genes, pprI and pprA, couldn't confer tolerance. PprI protein is known as a inducer to genes expression such as recA and pprA. Therefore, expression of only pprI may be ineffective for cell survival. Moreover, inserting heavy gene often causes decline of cell survival.</p><br />
<p>PprA protein has a function for repairing DNA damaged with blunt end. UV exposure causes thymine dimer, not related to blunt end. We suggest that pprA gene may have no function for repairing DNA damaged by UV but some repairing function for other types of damage such as by chemicals, of cause, radiation.</p><br />
<p>Fortunately, our result about gene mix(connected each genes) showed high tolerance to UV exposure.</p><br />
<p>RecA gene could induce high cell viability. RecA protein has key role of SOS response. <br />
This result revealed that the cell inserted recA gene can get tolerance against DNA damage. </p><br />
<br />
<br />
<br />
===SOS promoter assay===<br />
<p>We assayed the RecA promoter ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]) by attaching a lycopene biosynthesis gene cluster ([http://partsregistry.org/Part:BBa_K274100 K274100]) downstream as a reporter to yield the DNA damage detection device ([http://partsregistry.org/Part:BBa_K602013 K602013]). <br />
Transformed ''E. coli'' was exposed to UV light, and then, incubated for 2 hours. Lycopene as a reporter was extracted from cells with acetone. Please check [https://2011.igem.org/Team:Osaka/Protocols Protocol] for details.</p><br />
[[File:SOS_promoter_response.png|450px|center]]<br />
<br />
<p>Response is defined as absorbance at 474nm (peak absorbance for lycopene) divided by OD600, followed by subtraction of background (non-irradiated samples) absorbance values.</p><br />
<p>We observed a response to UV irradiation that increased with energy dosage from 200 to 400 J/m^2. Response decreased with higher energy dosages, perhaps as a result of intensive DNA damage rendering lycopene biosynthesis genes non-functional.</p><br />
<br />
=== Future work ===<br />
<p>'''Cell viability'''</p><br />
<p>We created some parts (PprI , PprA , PprM , RecA) but did not have enough time to evaluate them completely.</p><br />
<p>The effects of each parts to cell viability are shown above, but we will be able to evaluate them more precisely by measuring the effects of other combined parts. (We have already assayed a combined part, mix(all four parts combined).)</p><br />
<p>We should test responses of cells to other damage types, too.</p><br />
<p>'''SOS response'''</p><br />
<p>Our project is "Bio-dosimeter", so we should construct two devices about damage tolerance and damage detection respectively.</p><br />
<p>Our constructed bio-dosimeter shows the level of radiation by one pigment, but it's somewhat difficult to identify the level from light and shade of one color.Therefore, in future, Bio-dosimeter should be going to show the level of radiation using several pigments and promoters.</p><br />
<br />
=== References ===<br />
<ol><br />
<br />
<li>放射線抵抗性細菌の新規DNA修復促進タンパク質 , 佐藤勝也 その他 (2006)(Japanese books)</li><br />
<li>PprA: a protein implicated in radioresistance of ''Deinococcus radiodurans'' stimulates catalase activity in ''Escherichia coli'', Swathi Kota et al (2006)</li><br />
</ol><br />
</div></div>Shaohttp://2011.igem.org/File:2011_osaka_tolerance_2.pngFile:2011 osaka tolerance 2.png2011-10-28T18:58:41Z<p>Shao: </p>
<hr />
<div></div>Shaohttp://2011.igem.org/File:2011_osaka_tolerance_1.pngFile:2011 osaka tolerance 1.png2011-10-28T18:58:12Z<p>Shao: </p>
<hr />
<div></div>Shao