Team:UT-Tokyo/Project/Discussion

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=Discussion=
=Discussion=
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== TBD ==
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:Our experiments demonstrated that ''cheZ''-mediated motility regulation is possible and Asp regulates ''E. coli'' migration. Evaluation of Asp-production system is incomplete due to limited means to quantitate Asp, but our simulation demonstrated that an increase in bacterial concentration can be achieved using our system and that this increase in concentration will raise the efficiency of bioremediation.
 +
=Chemoattraction=
 +
:We have decided to construct a system in which an Asp-gradient forms around ''E. coli'' cells responding to the stimulus. To realize this system, we checked the feasibility of two steps, Asp Taxis, and Asp Production.
 +
 +
==Asp Taxis==
 +
:Here we discuss the ''E. coli'' chemotaxis toward introduced aspartate. We measured the diffusion of the colonies to find out how far the cells move by the chemotaxis. (See Assay 1) We will consider whether this observed result was compatible with a previous study.
 +
 +
:First, we consider the Asp-gradient generated when an aspartate solution was instilled. To visualize the generated Asp-gradient, we organized an experiment in which we measured the aspartate concentration at various time points and distances from the instillation point. As a result, we obtained the time and distance dependency of the aspartate diffusion. (See Assay 1) On the other hand, based on a previous study, we estimated the diffusion coefficient from the molecular mass of aspartate and solved the diffusion equation to simulate the theoretical transition of the aspartate concentrations across the plate. (See model 1)
 +
 +
:Next, we focus on the sensitivity of the cells to aspartate. Our experimental result showed that ''E. coli'' located 25mm away from the aspartate instillation point (10 mM aspartate solution 40 uL) was attracted to the aspartate, a point that the former simulation predicted the aspartate concentration to be 0.1 uM = 10<html><sup>-4</sup></html> M.
 +
 +
:According to a previous study, ''E. coli'' can detect aspartate at concentrations of 10<html><sup>-8</sup></html> M to 10<html><sup>-2</sup></html> M, especially if it is higher than 10<html><sup>-5</sup></html> M, and swim toward the higher aspartate concentration <html><sup class="ref">[1]</sup></html>. Our experimental result was not incompatible with these results.
 +
 +
==Asp Production==
 +
:Next we discuss the production of aspartate generating ''E. coli''. In the Asp Taxis part, we showed that we can predict the required amount of aspartate for chemotaxis. To confirm whether aspA+ cells can synthesize enough aspartate, we tried to analyze the quantity of synthesized aspartate by HPLC. However, we could not afford this method and our system is left to be verified.
 +
:The previous study showed that a 35-ml reaction solution and 50 mg of Dry Cell Weight of free cells at 37°C with constant shaking could efficiently produce L-Asp and it reached a saturated level (583 mM) within 1 h.<html><sup class="ref">[3]</sup></html> This suggests that our cells would generate 10<html><sup>-17</sup></html> mol/sec/cell aspartate. Our Asp Production experiment was conducted using a similar construct. So we assumed that in a circle with a radius of 5 mm on the plate (the same size of the introduced drop of our Asp Taxis Assay) they would produce 40 nmol of aspartate (the same amount as the introduced amount of our Asp Taxis Assay) in 1 hour. This assumption requires 1.1*10<html><sup>6</sup></html> cells/hr which amounts to 9 uL cell volume of OD=1.0<html><sup class="ref">[4]</sup></html>.  This result suggests that our self-mustering system is realistic. Therefore by overexpression of AspA, it is possible to generate the Asp gradient sufficient to gather nearby ''E. coli''.
 +
 +
:In conclusion, these discussions suggest that our self-mustering system can be realized. By using the proper aspartate-analytical method, HPLC, we will be able to verify our Asp production system and accomplish our goal, self-mustering system.
 +
=Cell Arrest System using Motility Control=
 +
:''E. coli'' natively have a property called chemotaxis, which is achieved by regulating flagellar movement. ''E. coli'' swim straight when their flagella rotate counter-clockwise, and they tumble when their flagella rotate clockwise. ''cheZ''  is one of the flagellar-regulating genes, and CheZ dephosphorylates CheY. Flagellum of the cell rotates clockwise when CheY is phosphorylated, and its dephosphorylation leads the flagellum to rotate counter-clockwise and makes the cell to go straight. In our project we aim to arrest E.coli near the substrate area by the motility regulation through CheZ manipulation.
 +
 +
:To evaluate the motility of CheZ- strain transformed with a IPTG-inducible CheZ expression device (BBa_K518006), we measured the colony size and the whiteness index (WI) 24, 48, and 72 hours after IPTG-induction. The colony size of BBa_K518006-transformed bacteria was significantly larger than that of an untransformed CheZ- strain. These results suggest that IPTG-induced CheZ expression rescues the cell motility of CheZ- strain.
 +
 +
:Second, the IPTG-inducible CheZ repression device (BBa_K518008) was transformed to ''E. coli'' and plated in the presence of IPTG. The size of these colonies were smaller than cells expressing ''cheZ''. This shows that the motility of these cells decrease in the presence of IPTG.
 +
 +
:The result of our computer simulation suggests that cell arrest system enhances the efficiency of remediation, indicating that our system is effective.
 +
=Why We Chose to Work with SOS Promoters=
 +
 +
:As described in the project overview, our system works by combining and adding the adequate induction apparatus, including receptors, transcription factors and promoters. The SOS response is a rapid reaction in microorganisms induced by DNA damage. We used SOS promoters to control the induction of our system as proof of principle that components of our system are functional.
 +
 +
:Transcription by SOS promoters is negatively regulated by a native repressor of ''E. coli'', LexA. The induction intensity of a SOS promoter is considered to be determined by the affinity of LexA. In our research we use ''sulAp'' because it is usually strongly repressed but is efficiently induced by UV irradiation. The measurement of ''sulAp'' by dual luciferase assay is described in the Results page in detail.
 +
 +
:The selection of other pairs of receptors and substrates allows you to make it possible to improve existing and new bioremediation systems. Our results concerning the UV switch also offer reliable data to other projects involving bioremediation.
 +
 +
 +
<html>
 +
<h2>References</h2>
 +
<div id="references">
 +
<ul>
 +
  <li id="ref_1">[1] ROBERT MESIBOV AND JULIUS ADLER (1972) Chemotaxis Toward Amino Acids in　Escherichia coli. JOURNAL OF BACTERIOLOGY, 112(1), 315-326.</li>
 +
  <li id="ref_2">[2] VICTOR  JOHN  HARDING  AND  FRANCIS  H.  S.  WARNEFORD. (1916) THE  NINHYDRIN  REACTION  WITH  AMINO-ACIDS  AND AMMONIUM  SALTS.</li>
 +
  <li id="ref_3">[3] Yun-Peng Chao, Tsuey-Er Lo Neng-Shing Luo (2000) Selective production of L-aspertic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli. Enzyme and Microbial Technology, 27, 19-25</li>
 +
  <li id="ref_4">[4] E.coli statistics http://www.ccdb.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi</li>
 +
</ul>
 +
</div>
 +
</html>
{{:Team:UT-Tokyo/Templates/EndContent}}
{{:Team:UT-Tokyo/Templates/EndContent}}

Latest revision as of 01:59, 6 October 2011

Discussion

Our experiments demonstrated that cheZ-mediated motility regulation is possible and Asp regulates E. coli migration. Evaluation of Asp-production system is incomplete due to limited means to quantitate Asp, but our simulation demonstrated that an increase in bacterial concentration can be achieved using our system and that this increase in concentration will raise the efficiency of bioremediation.

Chemoattraction

We have decided to construct a system in which an Asp-gradient forms around E. coli cells responding to the stimulus. To realize this system, we checked the feasibility of two steps, Asp Taxis, and Asp Production.

Asp Taxis

Here we discuss the E. coli chemotaxis toward introduced aspartate. We measured the diffusion of the colonies to find out how far the cells move by the chemotaxis. (See Assay 1) We will consider whether this observed result was compatible with a previous study.
First, we consider the Asp-gradient generated when an aspartate solution was instilled. To visualize the generated Asp-gradient, we organized an experiment in which we measured the aspartate concentration at various time points and distances from the instillation point. As a result, we obtained the time and distance dependency of the aspartate diffusion. (See Assay 1) On the other hand, based on a previous study, we estimated the diffusion coefficient from the molecular mass of aspartate and solved the diffusion equation to simulate the theoretical transition of the aspartate concentrations across the plate. (See model 1)
Next, we focus on the sensitivity of the cells to aspartate. Our experimental result showed that E. coli located 25mm away from the aspartate instillation point (10 mM aspartate solution 40 uL) was attracted to the aspartate, a point that the former simulation predicted the aspartate concentration to be 0.1 uM = 10-4 M.
According to a previous study, E. coli can detect aspartate at concentrations of 10-8 M to 10-2 M, especially if it is higher than 10-5 M, and swim toward the higher aspartate concentration [1]. Our experimental result was not incompatible with these results.

Asp Production

Next we discuss the production of aspartate generating E. coli. In the Asp Taxis part, we showed that we can predict the required amount of aspartate for chemotaxis. To confirm whether aspA+ cells can synthesize enough aspartate, we tried to analyze the quantity of synthesized aspartate by HPLC. However, we could not afford this method and our system is left to be verified.
The previous study showed that a 35-ml reaction solution and 50 mg of Dry Cell Weight of free cells at 37°C with constant shaking could efficiently produce L-Asp and it reached a saturated level (583 mM) within 1 h.[3] This suggests that our cells would generate 10-17 mol/sec/cell aspartate. Our Asp Production experiment was conducted using a similar construct. So we assumed that in a circle with a radius of 5 mm on the plate (the same size of the introduced drop of our Asp Taxis Assay) they would produce 40 nmol of aspartate (the same amount as the introduced amount of our Asp Taxis Assay) in 1 hour. This assumption requires 1.1*106 cells/hr which amounts to 9 uL cell volume of OD=1.0[4]. This result suggests that our self-mustering system is realistic. Therefore by overexpression of AspA, it is possible to generate the Asp gradient sufficient to gather nearby E. coli.
In conclusion, these discussions suggest that our self-mustering system can be realized. By using the proper aspartate-analytical method, HPLC, we will be able to verify our Asp production system and accomplish our goal, self-mustering system.

Cell Arrest System using Motility Control

E. coli natively have a property called chemotaxis, which is achieved by regulating flagellar movement. E. coli swim straight when their flagella rotate counter-clockwise, and they tumble when their flagella rotate clockwise. cheZ is one of the flagellar-regulating genes, and CheZ dephosphorylates CheY. Flagellum of the cell rotates clockwise when CheY is phosphorylated, and its dephosphorylation leads the flagellum to rotate counter-clockwise and makes the cell to go straight. In our project we aim to arrest E.coli near the substrate area by the motility regulation through CheZ manipulation.
To evaluate the motility of CheZ- strain transformed with a IPTG-inducible CheZ expression device (BBa_K518006), we measured the colony size and the whiteness index (WI) 24, 48, and 72 hours after IPTG-induction. The colony size of BBa_K518006-transformed bacteria was significantly larger than that of an untransformed CheZ- strain. These results suggest that IPTG-induced CheZ expression rescues the cell motility of CheZ- strain.
Second, the IPTG-inducible CheZ repression device (BBa_K518008) was transformed to E. coli and plated in the presence of IPTG. The size of these colonies were smaller than cells expressing cheZ. This shows that the motility of these cells decrease in the presence of IPTG.
The result of our computer simulation suggests that cell arrest system enhances the efficiency of remediation, indicating that our system is effective.

Why We Chose to Work with SOS Promoters

As described in the project overview, our system works by combining and adding the adequate induction apparatus, including receptors, transcription factors and promoters. The SOS response is a rapid reaction in microorganisms induced by DNA damage. We used SOS promoters to control the induction of our system as proof of principle that components of our system are functional.
Transcription by SOS promoters is negatively regulated by a native repressor of E. coli, LexA. The induction intensity of a SOS promoter is considered to be determined by the affinity of LexA. In our research we use sulAp because it is usually strongly repressed but is efficiently induced by UV irradiation. The measurement of sulAp by dual luciferase assay is described in the Results page in detail.
The selection of other pairs of receptors and substrates allows you to make it possible to improve existing and new bioremediation systems. Our results concerning the UV switch also offer reliable data to other projects involving bioremediation.


References

  • [1] ROBERT MESIBOV AND JULIUS ADLER (1972) Chemotaxis Toward Amino Acids in　Escherichia coli. JOURNAL OF BACTERIOLOGY, 112(1), 315-326.
  • [2] VICTOR JOHN HARDING AND FRANCIS H. S. WARNEFORD. (1916) THE NINHYDRIN REACTION WITH AMINO-ACIDS AND AMMONIUM SALTS.
  • [3] Yun-Peng Chao, Tsuey-Er Lo Neng-Shing Luo (2000) Selective production of L-aspertic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli. Enzyme and Microbial Technology, 27, 19-25
  • [4] E.coli statistics http://www.ccdb.ualberta.ca/CCDB/cgi-bin/STAT_NEW.cgi

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