Difference between revisions of "Part:BBa K602008:Experience"

Revision as of 01:36, 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 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.

Mitomycin C tolerance

The D. radiodurans proteins were also tested for their ability to confer resistance to Mitomycin C, a potent drug that causes DNA crosslinking and double-strand breaks. Transformed, pre-cultured cells were induced with IPTG, exposed to 2µg/ml Mitomycin C for 2h then plated on agar 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.

PprM and RecA were effective in conferring tolerance. Induced PprI and PprA parts did not significantly increase tolerance relative to the wild type. As UV irradiation and Mitomycin C exposure cause different types of DNA damage (UV causes pyridine dimerizations while Mitomycin C can cause double strand breaks) we conclude that PprM and RecA may have the ability to repair a wider range of DNA damage than PprI or PprA.

User Reviews

UNIQ1881014fb0edb0ac-partinfo-00000000-QINU UNIQ1881014fb0edb0ac-partinfo-00000001-QINU

@@ Line 3: / Line 3: @@
 how you used this part and how it worked out.
-===Applications of BBa_K602008===
+=== 2011 Osaka ===
-==== 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 and pre-cultured cells were 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.
+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.
-[[Image:2011_osaka_tolerance.png|600px]]
+[[Image:IPTG induction effects.png|800px]]
-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.
+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.
-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 a positive note, both RecA and the combined part (comprising all four proteins) significantly increased UV tolerance.
+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.
+==== Mitomycin C tolerance ====
+The <i>D. radiodurans</i> proteins were also tested for their ability to confer resistance to Mitomycin C, a potent drug that causes DNA crosslinking and double-strand breaks. Transformed, pre-cultured cells were induced with IPTG, exposed to 2&micro;g/ml Mitomycin C for 2h then plated on agar 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.
+[[Image:Viability_mitomycin.png|300px]]
+PprM and RecA were effective in conferring tolerance. Induced PprI and PprA parts did not significantly increase  tolerance relative to the wild type. As UV irradiation and Mitomycin C exposure cause different types of DNA damage (UV causes pyridine dimerizations while Mitomycin C can cause double strand breaks) we conclude that PprM and RecA may have the ability to repair a wider range of DNA damage than PprI or PprA.
 ===User Reviews===