Team:Osaka/Tests

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Latest revision as of 14:51, 21 October 2012

Tests

Damage tolerance assay

Radioresistance parts contain codon rarely used in E.coli. Codon optimization and a better expression system are needed to make our parts functional, so we transformed plasmid DNA into E.coli Rosetta.
To measure the DNA damage tolerance conferred by each part, 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. For detailed protocols, refer to the Protocols page.

The tolerance parts tested were as follows:

Parts containing one gene each

CDS: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K602005 PprI], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K602006 PprA], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K602007 PprM] or [http://partsregistry.org/wiki/index.php?title=Part:BBa_K602008 RecA]

Parts containing two genes

CDS1+2: [http://partsregistry.org/wiki/index.php?title=Part:BBa_K602016 PprI+RecA], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K602017 PprA+RecA], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K602020 PprM+RecA], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K602015 PprI+PprA],[http://partsregistry.org/wiki/index.php?title=Part:BBa_K602018 PprI+PprM], [http://partsregistry.org/wiki/index.php?title=Part:BBa_K602019 PprA+PprM],

Discussion

Single-gene parts

Against Mitomycine C

We found that

PprI did not increase the tolerance alone. PprI is a pleiotropic gene, which senses DNA lesions, regulates the downstream repair genes and also carries catalase activity. 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 interstrand crosslinks and single strand breaks induced by Mitomycin C.
In our previous experiment using UV irradiation, PprM showed to confer tolerance slightly. Though it is said that PprM is a modulator of the PprI-dependent damage response that depends on downstream effector such as PprA, our results indicates that it is protective for E.coli by itself.
In our previous experiments, RecA of D. radiodurans conferred the highest tolerance among the four radiotolerance genes (against UV irradiation). We showed that RecA much increased tolerance against DNA damaging agents, too. Maybe, it supports the effect of E.coli's native RecA each other.

Against Hydrogen peroxide

Actually, 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

Two-gene combinations

Against Mitomycine C

While PprI alone was unable to confer the tolerance to this agent, the combination of PprI and PprA somehow increased the tolerance. This is in accordance with the role of PprI as an inducer of PprA.
The fact that this combination much increased the tolerance, agrees with the role of PprI as an inducer of D. radiodurans RecA.
Though it is said that PprM is not a modulator of RecA, here we shiowed 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.

Against Hydrogen peroxide

Though PprA or PprM alone did not increase tolerance, the combination of PprI and other radiotolerance genes somehow improved the tolerance. This agrees with the role of PprI as an enhancer of enzyme activities of catalases.

Conclusion

In order to render E. coli tolrerant to DNA lesions, we tested possible exogenous radioresistance genes originally isolated from D.radiodurance.

First, PprM appears to protect against to interstrand crosslinks and/or single strand breaks induced by Mitomycin C. 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.
In addition, our results indicated that PprI, as a global regulator of the DNA repair system, alone does not secure against Mitomycin C and UV irradiation. This is in contradiction to a previous report that PprI confers radiotolerance to E. coli.
Finally, D. radiodurans's RecA confer resistance to any mutagen despite past reports that this protein is lethal to E. coli.

Promoter assay

This is our goal of construct to detect and report the DNA damages. This construct needs responsive promoter to sense DNA damage and also needs reporter genes to produce a sort of color pigments.
Last year, 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/wiki/index.php?title=Part:BBa_K274100 K274100]) downstream as a reporter. This construct successfully produce lycopene in response to DNA damage. However, it remains unclear in the quantitative relation between promoter activity and color intensity. In order to evaluate the promoter activity quantitatively and accurately, we also employed the Dual Luciferase Reporter Assay System as a control. Transformed E. coli was exposed to DNA damaging agents and then incubated for 2 hours. For details check the Protocols page.

We are trying to construct this promoter evaluation device utilizing firefly and renilla luciferase, but not yet finished.

Future work

1, Complete the Dual Luciferase Assay System

We are trying to assemble the Dual Luciferase Assay System from existing Biobrick parts, but have not finished it yet. So, we will go on to complete it.

2, Quantification of damages to "Bio-dosimeter"

One of the weak point of our "Bio-dosimeter" is difficulty to quantify the damages, so that, to quantify radiation. Therefore, for practical use, we have to evaluate the ovserved output and compare the dosed danage source to quantify the measurement by the "Bio-dosimeter".

@@ Line 4: / Line 4: @@
 == Tests ==
 === Damage tolerance assay ===
-Radioresistance parts contain codon rarely used in <i>E.coli</i>. Codon optimization and a better expression system are needed to make our parts functional, so we transformed plasmid DNA into <i>E.coli</i> Rosetta.
+Radioresistance parts contain codon rarely used in <i>''E.coli''</i>. Codon optimization and a better expression system are needed to make our parts functional, so we transformed plasmid DNA into <i>''E.coli''</i> Rosetta.
 <br>
-To measure the DNA damage tolerance conferred by each part, we used DNA damaging agents (such as Mitomycin C and Hydrogen peroxide) as a source of DNA damage and then assayed the survival rates. Transformed ''E. coli'' was exposed to DNA damaging agents and then incubated for 2 hours. Cells were plated on agar plates at different dilutions and air dried. 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://2012.igem.org/Team:Osaka/Protocols Protocols page].
+To measure the DNA damage tolerance conferred by each part, 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&deg;C. For detailed protocols, refer to the [https://2012.igem.org/Team:Osaka/Protocols Protocols page].
 The tolerance parts tested were as follows:
@@ Line 25: / Line 25: @@
 ==== Discussion ====
 ===== Single-gene parts =====
-Mitomycine C
+Against Mitomycine C
-* PprI did not appear to increase tolerance, corroborating with its known role as an inducer of other radiotolerance proteins. As <i>E. coli</i> would lack these required <i>D. radiodurans</i> proteins it is expected that PprI would not be able to confer tolerance on its own.
-* PprA which repairs blunt-ended breaks, also did not seem to confer tolerance. PprA may not protect against interstrand crosslinks and single strand breaks induced by Mitomycin C
-* Perhaps the most unexpected result was that from PprM. In our previous experiment (using UV irradiation), PprM also appeared to confer a degree of tolerance although it was not significant enough to be of note. 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.
-* <i>D. radiodurans</i> RecA was indicated in our previous experimental results to confer the highest tolerance among the four radiotolerance genes (using UV irradiation). Again this time, RecA raised the highest tolewrance to <i>E. coli</i>. This might be because it plays a complementary role to native <i>E. coli</i> RecA.
-Hydrogen peroxide
-* Either pprA or PprM actually decreased tolerance. It is possible that expression of PprA or PprM would not do much to improve cell survival and may actually increase the burden on the cell.
-* However, both PprI and RecA significantly increased tolerance, which agrees with the role of PprI as an enhancer of enzyme activities of catalases
+We found that
+* PprI did not increase the tolerance alone. PprI is a pleiotropic gene, which senses DNA lesions, regulates the downstream repair genes and also carries catalase activity. As <i>''E. coli''</i> lacks downstream proteins of <i>D. radiodurans</i>, 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 interstrand crosslinks and single strand breaks induced by Mitomycin C.
+* In our previous experiment using UV irradiation, PprM showed to confer tolerance slightly. Though it is said that PprM is a modulator of the PprI-dependent damage response that depends on downstream effector such as PprA, our results indicates that it is protective for <i>''E.coli''</i> by itself.
+* In our previous experiments, RecA of <i>''D. radiodurans''</i> conferred the highest tolerance among the four radiotolerance genes (against UV irradiation). We showed that RecA much increased tolerance against DNA damaging agents, too. Maybe, it supports the effect of <i>''E.coli''</i>'s native RecA each other.
+Against Hydrogen peroxide
+* Actually, 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
 ===== Two-gene combinations =====
-Mitomycine C
+Against Mitomycine C
-*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.
+*While PprI alone was unable to confer the tolerance to this agent, the combination of PprI and PprA somehow increased the tolerance. This is in accordance with the role of PprI as an inducer of PprA.
-*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.
+*The fact that this combination much increased the tolerance, agrees with the role of PprI as an inducer of <i>D. radiodurans</i> RecA.
-* 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 <i>E. coli</i> that benefit from the presence of PprM
+* Though it is said that PprM is not a modulator of RecA, here we shiowed 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 <i>E. coli</i> that benefit from the presence of PprM.
-Hydrogen peroxide
-* Though PprA or PprM alone did not increase any tolerance, the combination of PprI and any other radiotolerance genes worked to some degree. This agrees with the role of PprI as an enhancer of enzyme activities of catalases.
+Against Hydrogen peroxide
+* Though PprA or PprM alone did not increase tolerance, the combination of PprI and other radiotolerance genes somehow improved the tolerance. This agrees with the role of PprI as an enhancer of enzyme activities of catalases.
 =====  Conclusion =====
-We have obtained several interesting results from our DNA damage tolerance assays.
+In order to render <i>''E. coli''</i> tolrerant to DNA lesions, we tested possible exogenous radioresistance genes originally isolated from <i>''D.radiodurance''</i>.
-First, PprM appears to protect against to interstrand crosslinks and single strand breaks induced by Mitomycin C. 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 <i>E. coli</i> genes to advantageous effect.
-In addition, our results indicated that PprI, as a global regulator of the DNA repair system, alone does not secure against . 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.
-Finally, we have shown that PprA, when expressed in sufficient quantities, does appear to confer tolerance to E. coli.
-・MMC→DNA鎖に結合した後、分解され、ラジカル化。DNA鎖切断や架橋形成を引き起こす(遺伝情報を特異的に破壊する。)
-・H2O 2→それ自体が反応性が高く、E.coliのあらゆる組織にダメージを与える。(これは、MMCが遺伝情報を特異的に破壊するのと対照的)
-仮説
-以上の前提を踏まえて、H2O2を用いたTolerance　assayでE.coliの生存率を左右したのはH2O2がE.ciliのDNAにダメージをあたえたからというよりも、むしろH2O2がE.coliの諸組織を酸化し、生命機能を維持できなくさせたためだった可能性がある。
-根拠
-・MMCとH2O2の間の作用機構の相違
-・H2O2を用いたTolerance assay　で、(Catalasisを活性化する)PprIをいれたすべてのE.coliについて耐性の上昇がみられた。
-・MMCをもちいたassayにおいて耐性の上昇がみられたPprM、RecA+PprM、PprM+PprA導入体についてをH2O2用いたassayでは耐性の上昇がみられなかったこと。
-→BUT、H2O2を用いたassayにおけるRecA　singleでの耐性の上昇が説明不可
+First, PprM appears to protect against to interstrand crosslinks and/or single strand breaks induced by Mitomycin C. 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 <i>E. coli</i> genes to advantageous effect.
+<br>
+In addition, our results indicated that PprI, as a global regulator of the DNA repair system, alone does not secure against Mitomycin C and UV irradiation. This is in contradiction to a previous report that PprI confers radiotolerance to <i>''E. coli''</i>.
+<br>
+Finally, <i>''D. radiodurans''</i>'s RecA confer resistance to any mutagen despite past reports that this protein is lethal to <i>E. coli</i>.
 === Promoter assay ===
-<p>We assayed the promoter of the SOS gene RecA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_J22106 J22106]) and sulA ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K518010 K518010]).
+[[File:Construction.png|300px]]
-To measure the DNA damage detection, we used antibacterial agents as a source of DNA damage. To quantitatively and accurately evaluate the promoter activity, dual luciferase assay method was employed.
+<p>
-Transformed ''E. coli'' was exposed to antibacterial agents and then incubated for 2 hours. For details check the [https://2012.igem.org/Team:Osaka/Protocols Protocols page].</p>
+This is our goal of construct to detect and report the DNA damages. This construct needs responsive promoter to sense DNA damage and also needs reporter genes to produce a sort of color pigments.
+<br>
+Last year, 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/wiki/index.php?title=Part:BBa_K274100 K274100]) downstream as a reporter. This construct successfully produce lycopene in response to DNA damage. However, it remains unclear in the quantitative relation between promoter activity and color intensity. In order to evaluate the promoter activity quantitatively and accurately, we also employed the Dual Luciferase Reporter Assay System as a control. Transformed ''E. coli'' was exposed to DNA damaging agents and then incubated for 2 hours. For details check the [https://2012.igem.org/Team:Osaka/Protocols Protocols page].
+ </p>
 <html>
 <font size="3" color="#ff0000">
-  We were going to assemble the Dual Luciferase Assay System from existing Biobrick parts, but did not make it.
+  We are trying to construct this promoter evaluation device utilizing firefly and renilla luciferase, but not yet finished.
 </font>
 </html>
+=== Future work ===
+===== 1, Complete the Dual Luciferase Assay System =====
+<p>We are trying to assemble  the Dual Luciferase Assay System from existing Biobrick parts, but have not finished it yet. So, we will go on to complete it. </p>
+===== 2, Quantification of damages to "Bio-dosimeter"  =====
+<p>One of the weak point of our "Bio-dosimeter" is difficulty to quantify the damages, so that, to quantify radiation. Therefore, for practical use, we have to evaluate the ovserved output and compare the dosed danage source to quantify the measurement by the "Bio-dosimeter".</p>