Difference between revisions of "Part:BBa K602013:Experience"

(→‎Applications of BBa_K602013)
Line 24: Line 24:
 
<!-- End of the user review template -->
 
<!-- End of the user review template -->
 
<!-- DON'T DELETE --><partinfo>BBa_K602013 EndReviews</partinfo>
 
<!-- DON'T DELETE --><partinfo>BBa_K602013 EndReviews</partinfo>
 +
 +
=== 2012 Osaka ===
 +
 +
To measure the DNA damage tolerance conferred by each gene, we measured the survival rate in the presence of some DNA damaging agents (such as Mitomycin C and Hydrogen peroxide). Transformants E. coli were exposed to the DNA damaging agents and then incubated for 2 hours. Then, the cells were spreaded on agar plates at different dilutions. The plates were wrapped with aluminum foil and incubated in the dark. Colony-forming units were scored after 16h incubation at 37°C.
 +
 +
 +
====  Mitomycin C tolerance  ====
 +
 +
<html>
 +
<img src="https://static.igem.org/mediawiki/2012/thumb/e/e8/Viability_after_exposure_mitomycin_C.png/800px-Viability_after_exposure_mitomycin_C.png" width="800" height="300">
 +
</html>
 +
 +
 +
PprI did not increase the tolerance alone, while PprI regulates the multiple DNA repair and protection pathways in response to radiation. As E. coli lacks downstream proteins of D. radiodurans, it is expected that PprI would not be able to confer tolerance on its own.
 +
PprA, which repairs blunt-ended breaks in DNA lesions, also did not confer tolerance. PprA may not protect against DNA damages induced by MitomycinC.
 +
On the other hand, PprM and RecA much increased the tolerance.
 +
PprM is known to be a modulator of the PprI-dependent pathway. However, our data indicated that it is protective for E. coli by itself.
 +
 +
We observed increases in viability when certain tolerance genes were combined. For example, PprI by itself did not increase tolerance but boosted the ability of RecA to confer tolerance. This agrees with the role of PprI as an inducer of RecA in radiodurans.
 +
The combination of PprM and RecA also showed increase in tolerance. Since PprM is thought to not regulate RecA, this results indicate that PprM may induce or modulate other, unknown proteins and some of these proteins may have homologs in E. coli that benefit from the presence of PprM.
 +
 +
 +
====  Hydrogen peroxide tolerance  ====
 +
 +
<html>
 +
<img src="https://static.igem.org/mediawiki/2012/thumb/d/d2/Viability_after_exposure_hydrogen_peroxide.png/800px-Viability_after_exposure_hydrogen_peroxide.png" width="800" height="300">
 +
</html>
 +
 +
Each PprA and PprM alone decreased tolerance. It may be due to the cost of their expression in the stressful cellular environment at least.
 +
On the other hand, both PprI and RecA significantly increased tolerance, which agrees with the role of PprI as an enhancer of enzyme activities of catalases.
 +
 +
We observed increases in viability when certain tolerance genes were combined. PprA or PprM alone did not increase tolerance, the combination of PprI and other radiotolerance genes somehow improved the tolerance. This results confirms that PprI enhance the enzyme activities of catalases.

Revision as of 10:42, 6 October 2012

This experience page is provided so that any user may enter their experience using this part.
Please enter how you used this part and how it worked out.

Applications of BBa_K602013

2011 Osaka

This part was assayed for DNA damage detection ability as follows. E. coli transformed with this part was irradiated with UV light, and then incubated for 2 hours to provide sufficient time for lycopene production. Following that, the lycopene was extracted using acetone. The lycopene concentration was measured by absorbance at 474 nm and regarded as an approximate indicator of promoter activity.

In general, a high background level of lycopene was measured even in the absence of any irradiation. This might be due to basal expression from the SOS promoter which is needed for the SOS genes' rapid response to damage. Therefore, promoter response to UV irradiation was defined as the fractional increase of lycopene production over non-irradiated controls, for each irradiated sample/UV energy dosage.

2011 osaka promoter.png

User Reviews

UNIQab8ecb0655c8571f-partinfo-00000000-QINU UNIQab8ecb0655c8571f-partinfo-00000001-QINU

2012 Osaka

To measure the DNA damage tolerance conferred by each gene, we measured the survival rate in the presence of some DNA damaging agents (such as Mitomycin C and Hydrogen peroxide). Transformants E. coli were exposed to the DNA damaging agents and then incubated for 2 hours. Then, the cells were spreaded on agar plates at different dilutions. The plates were wrapped with aluminum foil and incubated in the dark. Colony-forming units were scored after 16h incubation at 37°C.


Mitomycin C tolerance


PprI did not increase the tolerance alone, while PprI regulates the multiple DNA repair and protection pathways in response to radiation. As E. coli lacks downstream proteins of D. radiodurans, it is expected that PprI would not be able to confer tolerance on its own. PprA, which repairs blunt-ended breaks in DNA lesions, also did not confer tolerance. PprA may not protect against DNA damages induced by MitomycinC. On the other hand, PprM and RecA much increased the tolerance. PprM is known to be a modulator of the PprI-dependent pathway. However, our data indicated that it is protective for E. coli by itself.

We observed increases in viability when certain tolerance genes were combined. For example, PprI by itself did not increase tolerance but boosted the ability of RecA to confer tolerance. This agrees with the role of PprI as an inducer of RecA in radiodurans. The combination of PprM and RecA also showed increase in tolerance. Since PprM is thought to not regulate RecA, this results indicate that PprM may induce or modulate other, unknown proteins and some of these proteins may have homologs in E. coli that benefit from the presence of PprM.


Hydrogen peroxide tolerance

Each PprA and PprM alone decreased tolerance. It may be due to the cost of their expression in the stressful cellular environment at least. On the other hand, both PprI and RecA significantly increased tolerance, which agrees with the role of PprI as an enhancer of enzyme activities of catalases.

We observed increases in viability when certain tolerance genes were combined. PprA or PprM alone did not increase tolerance, the combination of PprI and other radiotolerance genes somehow improved the tolerance. This results confirms that PprI enhance the enzyme activities of catalases.