Difference between revisions of "Part:BBa K602005:Experience"

Revision as of 00:06, 5 November 2011

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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 with other parts

Mitomycin C tolerance

User Reviews

UNIQ2e0eeb8af14e4589-partinfo-00000000-QINU UNIQ2e0eeb8af14e4589-partinfo-00000001-QINU

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 [[Image:IPTG induction effects.png|800px]]
-PprM and RecA
+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.
-It seemed that neither PprI nor PprA were capable of conferring any DNA damage tolerance in isolation. It is possible that expression of only PprI, a regulatory protein, would not do much to improve cell survival in the absence of it's downstream effectors while PprA, a DNA-binding protein mainly involved in repairing blunt-ended breaks in double-stranded DNA, may not protect against the thymine dimerization induced by UV. In fact, expression of either protein actually decreased tolerance, perhaps due to the increased burden on the cells.
-PprM did not have a clear positive or negative effect: although data seems to indicate increased survival at high (20 J/m^2) UV energy dosage, it was not significant enough to preclude experimental error.
+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.
-On a positive note, both RecA and the combined part (comprising all four proteins) significantly increased UV tolerance.
+It is interesting to note that in all cases, tolerance is increased upon IPTG induction, even in the wild type.
 ===== Combination with other parts =====