Difference between revisions of "Part:BBa K602020:Experience"
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | This experience page is provided so that any user may enter their experience using this part.<BR>Please enter | ||
how you used this part and how it worked out. | how you used this part and how it worked out. | ||
| − | === | + | === 2011 Osaka === |
| + | ==== UV tolerance ==== | ||
| + | Several <i>D. radiodurans</i> proteins related to DNA damage repair (PprI, PprM, PprA, RecA) were assayed for their ability to confer damage tolerance to host <i>E. coli</i> cells. Transformed, pre-cultured cells were induced with IPTG, plated and exposed to varying doses of UV radiation. Plates were then wrapped with aluminum foil to prevent further exposure and incubated for 16h. From colony counts, the survival percentages of irradiated samples relative to controls were calculated and used as an indicator of tolerance. | ||
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| + | [[Image:IPTG induction effects.png|800px]] | ||
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| + | Both PprM and RecA increased tolerance to UV irradiation even in the absence of IPTG induction, indicating that low levels of expression were sufficient for the function of these parts. The tolerance effect of PprM was unexpected given its role as a modulator of the PprI-dependent response mechanism in ''D. radiodurans''. This result indicates that PprM may regulate tolerance-related proteins endogenous to ''E. coli'' as well. | ||
| + | |||
| + | On the other hand, non-induced PprI and PprA actually ''decreased'' tolerance. While it is understandable that PprI, a global regulator of the DNA damage response, may not be able to confer tolerance in the absence of its usual downstream genes, it is not clear why the low level of expression expected in the absence of induction would result in decreased tolerance, although the metabolic burden caused by carrying the plasmid is suggested as one cause. | ||
| + | |||
| + | It is interesting to note that in all cases, tolerance is increased upon IPTG induction, even in the wild type. | ||
| + | |||
| + | ===== Combination of tolerance parts ===== | ||
| + | [[Image:Combined_parts_effect.png|400px]] | ||
| + | |||
| + | When composite parts combining two tolerance genes were constructed and tested (+IPTG induction), the combinations PprI+RecA and PprM+RecA showed significantly higher tolerance to UV irradiation. In particular PprI and RecA showed a high level of synergy, with the resultant tolerance being significantly higher than either gene alone. This agrees with the reported role of PprI as an inducer of RecA in ''D. radiodurans''. On the other hand the high tolerance conferred by PprM+RecA may be the additive effect of the two component genes. | ||
===User Reviews=== | ===User Reviews=== | ||
Revision as of 01:35, 5 November 2011
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.
2011 Osaka
UV tolerance
Several D. radiodurans proteins related to DNA damage repair (PprI, PprM, PprA, RecA) were assayed for their ability to confer damage tolerance to host E. coli cells. Transformed, pre-cultured cells were induced with IPTG, plated and exposed to varying doses of UV radiation. Plates were then wrapped with aluminum foil to prevent further exposure and incubated for 16h. From colony counts, the survival percentages of irradiated samples relative to controls were calculated and used as an indicator of tolerance.
Both PprM and RecA increased tolerance to UV irradiation even in the absence of IPTG induction, indicating that low levels of expression were sufficient for the function of these parts. The tolerance effect of PprM was unexpected given its role as a modulator of the PprI-dependent response mechanism in D. radiodurans. This result indicates that PprM may regulate tolerance-related proteins endogenous to E. coli as well.
On the other hand, non-induced PprI and PprA actually decreased tolerance. While it is understandable that PprI, a global regulator of the DNA damage response, may not be able to confer tolerance in the absence of its usual downstream genes, it is not clear why the low level of expression expected in the absence of induction would result in decreased tolerance, although the metabolic burden caused by carrying the plasmid is suggested as one cause.
It is interesting to note that in all cases, tolerance is increased upon IPTG induction, even in the wild type.
Combination of tolerance parts
When composite parts combining two tolerance genes were constructed and tested (+IPTG induction), the combinations PprI+RecA and PprM+RecA showed significantly higher tolerance to UV irradiation. In particular PprI and RecA showed a high level of synergy, with the resultant tolerance being significantly higher than either gene alone. This agrees with the reported role of PprI as an inducer of RecA in D. radiodurans. On the other hand the high tolerance conferred by PprM+RecA may be the additive effect of the two component genes.
User Reviews
UNIQfbdd80a622cd9d3c-partinfo-00000000-QINU UNIQfbdd80a622cd9d3c-partinfo-00000001-QINU