Team:Kyoto/Project/Goal C

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==Goal C: Characterization of the anti-killer gene==
==Goal C: Characterization of the anti-killer gene==
===Introduction===
===Introduction===
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As long as we know, there are two anti-killer gene against the killer gene,lysis cassette, S107 and SΔTMD1.
+
As long as we know, there are two anti-killer genes against the killer gene(lysis cassette), S107 and SΔTMD1.
S107 is expressed by S gene encoded by lysis cassette and SΔTMD1 is an S allele with the transmembrane domein 1(TMD1) deleted.
S107 is expressed by S gene encoded by lysis cassette and SΔTMD1 is an S allele with the transmembrane domein 1(TMD1) deleted.
Because TMD1 of S gene is essential for the function of lysis cassette as killer gene, SΔTMD1 inhibits lysis cassette more strongly than S107.([[Team:Kyoto/LearnMore#Lysis Cassette|Learn more]])
Because TMD1 of S gene is essential for the function of lysis cassette as killer gene, SΔTMD1 inhibits lysis cassette more strongly than S107.([[Team:Kyoto/LearnMore#Lysis Cassette|Learn more]])
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Then, we checked if SΔTMD1 prevents lysis cassette from causing the cell death.  
Then, we checked if SΔTMD1 prevents lysis cassette from causing the cell death.  
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To research this function of S&Delta;TMD1, we used the E.coli transformed with both these genes( <partinfo>BBa_K358021</partinfo>).
+
To investigate the function of S&DELTA;TMD1, we used the E.coli transformed with both killer and anti-killer genes ( <partinfo>BBa_K358021</partinfo>).
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Because E.coli is immediately dead if killer-gene is constitutively expressed, we made lysis cassette regulated by lac promoter. The E.coli bacteria were grown in the medium without IPTG, and after there were enough E.coli, they were cultured in the one with IPTG to express lysis cassette. After that, we checked, by measuring the absorbance (A550), if E.coli were alive due to S&Delta;TMD1 expressed constitutively.
+
Because E.coli will be killed immediately if the killer-gene is constitutively expressed, we constructed the lysis cassette regulated by lac promoter. At first, the E.coli bacteria were grown in the medium without IPTG. After the enough population of E.coli, they were cultured in the one with IPTG to express lysis cassette. After that, we checked the growth rate of E.coli by measuring the absorbance (A550) of the growth medium. Thus, we investigated the possibility whether E.coli that contains both the killer and anti-killer genes could alive due to the constitutive expression of S&DELTA;TMD1.  
===Method===
===Method===
====Bacterial strains====
====Bacterial strains====
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We used four types of E. coli, E. coli KRX transformed with <partinfo>BBa_K358021</partinfo>,  KRX transformed with <partinfo>BBa_K358022</partinfo> KRX transformed with <partinfo>BBa_K358024</partinfo> and KPX transfomed with  <partinfo>BBa_K358019</partinfo>.
+
We used four types of E. coli as follows: (1) E. coli KRX transformed with <partinfo>BBa_K358021</partinfo>,  (2)KRX transformed with <partinfo>BBa_K358022</partinfo>, (3)KRX transformed with <partinfo>BBa_K358024</partinfo> and (4)KRX transfomed with  <partinfo>BBa_K358019</partinfo>.
After this, we call each E.coli to abbreviate in order of name, LB2-1, lacLΔ, LB2-2 and lacL.
After this, we call each E.coli to abbreviate in order of name, LB2-1, lacLΔ, LB2-2 and lacL.
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*<partinfo>BBa_K358021</partinfo>:Anti-killergene is induced constitutively and killergene is regulated by lactose promoter.
*<partinfo>BBa_K358021</partinfo>:Anti-killergene is induced constitutively and killergene is regulated by lactose promoter.
It was used in experiment 2
It was used in experiment 2
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*<partinfo>BBa_K358022</partinfo>:lacP + SΔTMD1RRz, TMD1 is deleted from lysis cassette[SRRz].
+
*<partinfo>BBa_K358022</partinfo>:lacP + S&DELTA;TMD1RRz, TMD1 is deleted from lysis cassette[SRRz].
It was used in experiment 1  
It was used in experiment 1  
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*<partinfo>BBa_K358024</partinfo>: This part is the same to BBa_K358021, except for its constitutive promoter[BBa_J23101]. This part also induces constitutively SΔTMD1 gene, the anti-killer gene, and lambda lysis cassette[SRRz], the killer gene, is regulated by lacP.
+
*<partinfo>BBa_K358024</partinfo>: This part is the same to BBa_K358021, except for its constitutive promoter[BBa_J23101]. This part also induces constitutively S&DELTA;TMD1 gene, the anti-killer gene, and lambda lysis cassette[SRRz], the killer gene, is regulated by lacP.
It was used in the additional experiment, which is not finished.
It was used in the additional experiment, which is not finished.
*<partinfo>BBa_K358019</partinfo>: Lysis cassette regulated by lacP, Lysis cassette[SRRz] is regulated by lactose promoter.  
*<partinfo>BBa_K358019</partinfo>: Lysis cassette regulated by lacP, Lysis cassette[SRRz] is regulated by lactose promoter.  
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=====Experiment 2=====
=====Experiment 2=====
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We pick up three colonies from each plate, and cultivate them in the supplemented M9 medium overnight, about 16 hours. The overnight cultures were diluted to 0.1~0.11 in pre-warmed fresh the supplemented M9 medium. we measure A550 of the culture containing various IPTG at about 100 minute interval.
+
We pick up three colonies from each plate, and cultivate them in the supplemented M9 medium ~24-30 h (overnight).. The overnight cultures were diluted to 0.1~0.11 in pre-warmed fresh the supplemented M9 medium. we measure A550 of the culture containing various IPTG at about 100 minute interval.
===Result===
===Result===
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*less S&Delta;TMD1 proteins or more lysis cassette proteins was expressed than we expected.
*less S&Delta;TMD1 proteins or more lysis cassette proteins was expressed than we expected.
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=====Reason1:S&Delta;TMD1 in the plasmid did not worked correctly.=====
+
=====Reason1:S&Delta;TMD1 in the plasmid did not work correctly.=====
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To make sure the reason, we sequenced BBa_K358021 we used.  
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To investigate this reason, we sequenced BBa_K358021 we used.  
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It’s result showed that this construct is inserted into the plasmid  correctly, which demonstrated  that S&Delta;TMD1 worked correctly.
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The result showed that this construct is inserted into the plasmid  correctly, which demonstrated  that S&Delta;TMD1 worked correctly.
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So, first reason was not right.
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So, we assume that the first reason is negative.
=====Reason2:S&Delta;TMD1 is weaker anti-killer gene than we expected.=====
=====Reason2:S&Delta;TMD1 is weaker anti-killer gene than we expected.=====
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As anti-killer gene against lysis cassette, there are two genes, S107 and S&Delta;TMD1([[Team:Kyoto/LearnMore#Lysis Cassette|Learn more]]).
+
As anti-killer gene against lysis cassette, there are two genes, S107 and S&Delta;TMD1([[Team:Kyoto/LearnMore#Lysis Cassette|Learn more]]). .  
-
S107 is anti-killer gene, but because it has essential TMD1 of S for function of this killer gene, it’s function as anti-killer gene is weak compared with S&Delta;TMD1, which does not have TMD1 of ([[Team:Kyoto/LearnMore#Lysis Cassette|Learn more]]).
+
. S107 is the anti-killer gene. Because it has essential TMD1 of S for function of this killer gene, the anti-killer function of this gene is weak compared with S&Delta;TMD1, which does not have TMD1([[Team:Kyoto/LearnMore#Lysis Cassette|Learn more]]).
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So, we thought that S&Delta;TMD1 is a strong anti-killer gene, but from result of experiment 1 we doubted S&Delta;TMD1 was  a strong anti-killer gene. However, I could not make sure the reason.
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Thus, we expected that S&Delta;TMD1 is a strong anti-killer gene. However, from the result of experiment 2, we could not observe the function of S&Delta;TMD1 as a strong anti-killer gene under the tested condition.
-
 
+
=====Reason3: Expression balance between the anti-killer and killer genes was not optimized properly.=====
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=====Reason3: less S&Delta;TMD1 proteins or more lysis cassette proteins was expressed than we expected.=====
+
In order to investigate the possibility of this reason, we analyzed the expression level of the killer gene, lysis cassette and S&Delta;TMD1.   
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In order to make sure this reason, we researched the expression level of killer gene, lysis cassette and S&Delta;TMD1.   
+
As a report <sup>[[#RefL002|[2]]]</sup> says, it is necessary for the inhibition of lysis cassette that there are at least ~4.7 times more S107, anti-killer gene than lysis cassette proteins.  
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As a report <sup>[[#RefL002|[2]]]</sup> says, it is necessary for the inhibition of lysis cassette that there are at least 4.73 times more S107, anti-killer gene than lysis cassette proteins.  
+
So, Inhibition of this killer gene needs at least ~4.7 times more expression of the anti-killer gene than that of the killer gene. Thus, we calculated the ratio of the expression levels of the killer- and anti-killer- gene. The calculation was done under the assumptions that S gene, which lysis cassette includes, produces S105 and S107 at a 2.5:1 ratio([[Team:Kyoto/LearnMore#Lysis Cassette|Learn more]]), and that the strength of function of S&Delta;TMD1 as anti-killer gene is the same as that of S107. By this calculation, we knew that in LB-1, the anti-killer gene couldn't inhibit killer gene in the medium with more than 0.1246 mM IPTG. So, we assume that S&Delta;TMD1 was not expressed adequately to inhibit lysis cassette completly.  
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So, Inhibition of this killer gene needs at least 4.73 times more anti-killer gene than killer gene. Then, we calculated the ratio of killer gene and anti-killer gene, assuming that S gene, which lysis cassette includes, produces 2.5:1 S107 and S105([[Team:Kyoto/LearnMore#Lysis Cassette|Learn more]]), strength of S&DeltaTMD1 and S107 is the same. By this calcuration, we knew that in LB-1, the anti-killer gene couldn't inhibit killer gene in the medium with more than 0.1246mM IPTG. So, we insist that it is because S&Delta;TMD1 was not expressed adequately that S&Delta;TMD1 cannot inhibit lysis cassette completly.
+
We assume that our experimental results could be due to the 3rd reason (Reason 3) mentioned above, although we can not rule out the possibility of the second reason. However, it has been reported  <sup>[[#RefL001|[1]]]</sup> that S&Delta;TMD1 inhibits lysis cassette effectively. So, it is reasonable to assume that S&Delta;TMD1 exhibits enough activity to inhibit lysis cassette. That is why we think that the Reason 3 is most likely the answer. To confirm this possibility, we changed the promoter of S&Delta;TMD1 to stronger one. The experiment to check the function of S&Delta;TMD1 using the strong promoter is currently underway. Surely, this result will help us to understand why S&Delta;TMD1 could not function as we expected and to develop distinct cell-death controllable devices for the further studies.  
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+
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=====Conclusion=====
+
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We insist that the 3rd reason is right. Certainly, we have not made sure that 2nd Reason is not right. However, a report<sup>[[#RefL001|[1]]]</sup> says that S&Delta;TMD1 inhibits lysis cassette. So, it is natural that we think S&Delta;TMD1 have enough activity to inhibit lysis cassette.  
+
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That is why the 3rd reason is right.
+
-
 
+
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Then, in order to resolve this problem, we changed the promoter of S&Delta;TMD1 to stronger one, and now, are checking the function of S&Delta;TMD1. Surely, this result will help us discuss why S&Delta;TMD1 couldn’t function as we expected.
+
-
You must expect it!!
+
 +
====Conclusion====
 +
1) We made S&Delta;TMD1 by deleting TMD1 of Sgene
===Reference===
===Reference===
----
----
# <html><a name="RefL001"></a></html>Rebecca White, Tram Anh T. Tran, Chelsey A. Dankenbring, John Deaton, and Ry Young.2010.The N-Terminal Transmembrane Domain of _ S Is Required for Holin but Not Antiholin Function_. J. Bacteriol. 192: 725–733  
# <html><a name="RefL001"></a></html>Rebecca White, Tram Anh T. Tran, Chelsey A. Dankenbring, John Deaton, and Ry Young.2010.The N-Terminal Transmembrane Domain of _ S Is Required for Holin but Not Antiholin Function_. J. Bacteriol. 192: 725–733  
# <html><a name="RefL002"></a></html>CHUNG-YU CHANG, KIEBANG NAM, AND RY YOUNG1. 1995.2 S Gene Expression and the Timing of Lysis by Bacteriophage λ. J. Bacteriol. 177: 3283–3294
# <html><a name="RefL002"></a></html>CHUNG-YU CHANG, KIEBANG NAM, AND RY YOUNG1. 1995.2 S Gene Expression and the Timing of Lysis by Bacteriophage λ. J. Bacteriol. 177: 3283–3294

Revision as of 02:30, 28 October 2010

Contents

Goal C: Characterization of the anti-killer gene

Introduction

As long as we know, there are two anti-killer genes against the killer gene(lysis cassette), S107 and SΔTMD1. S107 is expressed by S gene encoded by lysis cassette and SΔTMD1 is an S allele with the transmembrane domein 1(TMD1) deleted. Because TMD1 of S gene is essential for the function of lysis cassette as killer gene, SΔTMD1 inhibits lysis cassette more strongly than S107.(Learn more) So, we selected SΔTMD1 as anti-killer gene.

Then, we checked if SΔTMD1 prevents lysis cassette from causing the cell death. To investigate the function of S&DELTA;TMD1, we used the E.coli transformed with both killer and anti-killer genes ( <partinfo>BBa_K358021</partinfo>). Because E.coli will be killed immediately if the killer-gene is constitutively expressed, we constructed the lysis cassette regulated by lac promoter. At first, the E.coli bacteria were grown in the medium without IPTG. After the enough population of E.coli, they were cultured in the one with IPTG to express lysis cassette. After that, we checked the growth rate of E.coli by measuring the absorbance (A550) of the growth medium. Thus, we investigated the possibility whether E.coli that contains both the killer and anti-killer genes could alive due to the constitutive expression of S&DELTA;TMD1.

Method

Bacterial strains

We used four types of E. coli as follows: (1) E. coli KRX transformed with <partinfo>BBa_K358021</partinfo>, (2)KRX transformed with <partinfo>BBa_K358022</partinfo>, (3)KRX transformed with <partinfo>BBa_K358024</partinfo> and (4)KRX transfomed with <partinfo>BBa_K358019</partinfo>. After this, we call each E.coli to abbreviate in order of name, LB2-1, lacLΔ, LB2-2 and lacL.

Construct

KyotoFigC001.pngKyotoFigC003.pngKyotoFigC002.pngKyotoFigC004.png

  • <partinfo>BBa_K358021</partinfo>:Anti-killergene is induced constitutively and killergene is regulated by lactose promoter.

It was used in experiment 2

  • <partinfo>BBa_K358022</partinfo>:lacP + S&DELTA;TMD1RRz, TMD1 is deleted from lysis cassette[SRRz].

It was used in experiment 1

  • <partinfo>BBa_K358024</partinfo>: This part is the same to BBa_K358021, except for its constitutive promoter[BBa_J23101]. This part also induces constitutively S&DELTA;TMD1 gene, the anti-killer gene, and lambda lysis cassette[SRRz], the killer gene, is regulated by lacP.

It was used in the additional experiment, which is not finished.

  • <partinfo>BBa_K358019</partinfo>: Lysis cassette regulated by lacP, Lysis cassette[SRRz] is regulated by lactose promoter.

Activating lactose promoter and expressing SRRz gene, it causes the cell lysis. Thus, lactose promoter must be repressed when this part is transformed. It was used in experiment 1 and 2.

Mesurement

Experiment 1

We pick up three colonies from each plate, and cultivate them in the supplemented M9 medium for a night, overnight. The overnight cultures were diluted to 0.1~0.11mM in pre-warmed fresh the supplemented M9 medium. 18h later, we measure A550 of the culture containing 0mM or 1mM IPTG.

Experiment 2

We pick up three colonies from each plate, and cultivate them in the supplemented M9 medium ~24-30 h (overnight).. The overnight cultures were diluted to 0.1~0.11 in pre-warmed fresh the supplemented M9 medium. we measure A550 of the culture containing various IPTG at about 100 minute interval.

Result

Experiment 1:Check the role of TMD1 of S gene

Fig.1.the comparion between lacL and lacLΔ.The black lines on bars' edges are error bar.

Then, in order to check if TMD1 of S gene is essential for this killer gene, we measured the cell number of lacLΔ, which does not have TMD1 of S gene and that is, encodes SΔTMD1 and that of lysis cassette. This result is below(Fig.1). From this result, after IPTG was added,the number of lacLΔ is not decreasing, while the number of lacLΔ is decresing. It proved that SΔTMD1 cannot function as killer gene and TMD1 is essential for this killer gene, lysis cassette. In addition, in the medium without IPTG, the number of lacLΔ is larger than that of lacLΔ.This supports a report [1]. which says that more proteins which is short are expressed than the ones which is long.

Experiment 2: Function check of SΔTMD1

We measured the cell number of lacLΔ at several time after induction by adding several concentration IPTG. The result is below (Fig.2). This result showed that in the medium with more than 0.03mM IPTG, the number of E. coli is decreasing at a certain point. It demonstrated that In at least more than 0.03mM IPTG, SΔTMD1 can't inhibit lysis cassette completly. Then, we measured the cell number of lacL in the same way, and compared the result of lacLΔ with that of lacL to check how SΔTMD1 inhibited lysis cassette(Fig.2). From this result, we knew in more than 0.5mM IPTG both Lacs and CTLA were decreasing in the number at a certain point, or CTLA was a little delayed decreaing in the number. This demonstrated that SΔTMD1 can't correctly inhibit lysis cassette. So, we doubted whether SΔTMD1 really functioned as anti-killer gene. Although SΔTMD1 has no TMD1 of S gene, SΔTMD1 couldn't show the function as anti-killer gene.


Fig.2.the result of function check of SΔTMD1. We measure A 550 changing IPTG concentration, 0, 0.02, 0.03, 0.05, 0.3, 0.5, 0.7, 1.0 (mM).
Fig.3.Comparison between lacL and LB2-1. We compared at IPTG concentration 0, 0.02, 0.03, 0.5, 1.0.

Discussion

TMD1 of S gene is essential for lysis cassette

The result of experiment 1 supports [[#RefL001|Ref.1]. That is, we were able to make sure that TMD1 of S gene is essential for lysis cassette as killer gene.

SΔTMD1 cannot inhibit lysis cassette completly

These results showed that SΔTMD1 cannot inhibit completely lysis cassette. This results can't supported a report on lysis [1]. which says that SΔTMD1 without TMD1 dominant-negatively inhibit lysis cassette. Why did we get the result against that report? Probably, there were three reasons below, we thought.

  • SΔTMD1 in the plasmid did not worked correctly.
  • S&DeltaTMD1 is weaker anti-killer gene than we expected.
  • less SΔTMD1 proteins or more lysis cassette proteins was expressed than we expected.
Reason1:SΔTMD1 in the plasmid did not work correctly.

To investigate this reason, we sequenced BBa_K358021 we used. The result showed that this construct is inserted into the plasmid correctly, which demonstrated that SΔTMD1 worked correctly. So, we assume that the first reason is negative.

Reason2:SΔTMD1 is weaker anti-killer gene than we expected.

As anti-killer gene against lysis cassette, there are two genes, S107 and SΔTMD1(Learn more). . . S107 is the anti-killer gene. Because it has essential TMD1 of S for function of this killer gene, the anti-killer function of this gene is weak compared with SΔTMD1, which does not have TMD1(Learn more). Thus, we expected that SΔTMD1 is a strong anti-killer gene. However, from the result of experiment 2, we could not observe the function of SΔTMD1 as a strong anti-killer gene under the tested condition.

Reason3: Expression balance between the anti-killer and killer genes was not optimized properly.

In order to investigate the possibility of this reason, we analyzed the expression level of the killer gene, lysis cassette and SΔTMD1. As a report [2] says, it is necessary for the inhibition of lysis cassette that there are at least ~4.7 times more S107, anti-killer gene than lysis cassette proteins. So, Inhibition of this killer gene needs at least ~4.7 times more expression of the anti-killer gene than that of the killer gene. Thus, we calculated the ratio of the expression levels of the killer- and anti-killer- gene. The calculation was done under the assumptions that S gene, which lysis cassette includes, produces S105 and S107 at a 2.5:1 ratio(Learn more), and that the strength of function of SΔTMD1 as anti-killer gene is the same as that of S107. By this calculation, we knew that in LB-1, the anti-killer gene couldn't inhibit killer gene in the medium with more than 0.1246 mM IPTG. So, we assume that SΔTMD1 was not expressed adequately to inhibit lysis cassette completly. We assume that our experimental results could be due to the 3rd reason (Reason 3) mentioned above, although we can not rule out the possibility of the second reason. However, it has been reported [1] that SΔTMD1 inhibits lysis cassette effectively. So, it is reasonable to assume that SΔTMD1 exhibits enough activity to inhibit lysis cassette. That is why we think that the Reason 3 is most likely the answer. To confirm this possibility, we changed the promoter of SΔTMD1 to stronger one. The experiment to check the function of SΔTMD1 using the strong promoter is currently underway. Surely, this result will help us to understand why SΔTMD1 could not function as we expected and to develop distinct cell-death controllable devices for the further studies.

Conclusion

1) We made SΔTMD1 by deleting TMD1 of Sgene

Reference


  1. Rebecca White, Tram Anh T. Tran, Chelsey A. Dankenbring, John Deaton, and Ry Young.2010.The N-Terminal Transmembrane Domain of _ S Is Required for Holin but Not Antiholin Function_. J. Bacteriol. 192: 725–733
  2. CHUNG-YU CHANG, KIEBANG NAM, AND RY YOUNG1. 1995.2 S Gene Expression and the Timing of Lysis by Bacteriophage λ. J. Bacteriol. 177: 3283–3294