Team:UT-Tokyo/Project/System

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=Project Overview=
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=System=
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Our project aims at improving the working efficiency of bioremediation by increasing bacterial density near the substrate. To do this, we developed two types of E. coli, “guiders” and “workers”.
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==Project Overview==
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The guiders are able to detect the substrate, and attract workers to themselves, where there is a high substrate concentration, when they do so.
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:As we stated in [[Team:UT-Tokyo/Project/Background|the Background page]], our SMART ''E.coli'' project aims at improving the working efficiency of bioremediation. To achieve this goal, we have designed the following two sub-systems. We first developed a Cell Assembling System, in which ''E. coli'' "guiders" detect a substrate and attract other cells to themselves via chemoattraction, therefore elevating the bacterial density at the substrate location. We constructed and tested an inducible chemoattractant (L-aspartate) producing device using an enzyme ''aspA''. Second, we devised a Cell Arrest System to prevent a time-dependent cell diffusion so that the elevated cell density is maintained. In this system, ''E. coli'' flagellum-regulating gene, ''cheZ'', was utilized to realize ''E.coli'' "workers" anchoring at the site of substrate.  By combining these two mechanisms, cell density at the location of substrate is expected to be greatly elevated and maintained, so that enhanced working effectiveness will be achieved.
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When the workers arrive around the substrate, they perform the bioremediation task assigned to them.
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The workers gather by chemoattraction and are then prevented from diffusing by a cell arrest mechanism, making the high density stable and last.
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=Chemotattraction=
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:To further reinforce the applicability of our SMART System, we also provided a "UV Switch", which enables us to switch on the SMART System even when a proper substrate-induced promoter does not exist. We investigated several UV-induced promoters, and successfully confirmed and characterized the UV-induced alteration of ''sulAp'' expression. Combining the UV Switch with our SMART System, it becomes possible to "switch on" the self-assembling and localization at the location of interest, which greatly enhances the future applications of this SMART System.
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E.coli natively have a property called 'chemotaxis,' and attracted to certain substances, 'chemoattractant.’ E.coli sense concentration gradient of the substances and advance in the direction where the concentration is higher.
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In this part we use chemoattraction to raise cell density. Specifically, we aim to realize substrate responsive chemoattrtactant overproduction and following cell assembly around the substrate.
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=Section1: Substrate-induced Cell Assembling System=
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:''E.coli'' have a native property called 'chemotaxis,' and are attracted to certain substances called 'chemoattractants.' ''E.coli'' detect and move towards a higher concentration of the substances.
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We have utilized L-Aspartate(L-Asp) as a chemoattractant. L-Asp is an amino acid verified as chemoattractant through preceding study<html><sup class="ref">[1]</sup></html> and our own experiment. By making E.coli discharge L-Asp, L-Asp gradient around the cell is generated and other E.coli (workers and other guiders) are attracted and gather toward the cell, and higher cell density is created around E.coli secreting L-Asp.
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:In our system we utilize this property to increase cell density. Specifically, we aim to realize substrate-responsive chemoattractant overproduction and a resultant cell assembly to the substrate.
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[[File:UT Tokyo Fumaric Acid.png|right]]
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Next, we have devised substrate responsive L-Asp overproduction mechanism. If guiders overproduce and secrete L-Asp in response to a particular substrate, they attract other bacteria and raise cell density around the substrate.  
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To make E.coli overproduce L-Asp when they sense a substrate, we attempt to insert the coding sequence of aspA in the downstream of a promoter responsive to the substrate.
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:{{:Team:UT-Tokyo/Templates/Image|file=UT Tokyo Fumaric Acid.png|caption=Figure. 1 Aspartate production catalyzed by AspA}}
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Pres - rbs - aspA - d.Ter
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:The chemoattractant we have utilized is aspartate (L-Asp). Aspartate has previously been shown to act as a chemoattractant<html><sup class="ref">[1]</sup></html>, which we confirmed by our own experiment, the cell diffusion assay. By making ''E.coli'' discharge aspartate, an aspartate concentration gradient around these bacteria is generated. Workers are attracted, creating a higher cell density around the aspartate-secreting ''E.coli''.
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The reaction of L-Asp production from fumaric acid and anmonium ion is catalyzed by aspartase(AspA) encoded by aspA<html><sup class="ref">[2]</sup></html>. (Figure. 1)
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:We have devised a substrate-responsive aspartate overproduction mechanism. By utilizing this mechanism, guiders overproduce and secrete aspartate in response to a particular substrate and attract other bacteria to enhance cell density around the substrate.
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The gene aspA is derived from E.coli and L-Asp is overproduced in the presence of enough AspA and the two substrates above.  
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:The following is the gene construct we designed.
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Once guiders sense a substrate and generate L-Asp gradient, other E.coli gather around the substrate as the result of chemoattraction. Among them, guiders attracted to the substrate spot also overproduce L-Asp if they sense the substrate. Here a positive feedback loop to create and keep gradient of L-Asp is formed. (Figure. 2)
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:[[File:UT_Tokyo_20-3-27-5.png|350px]]
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=Cell Arrest=
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:The coding sequence of AspA is inserted downstream of a substrate-responsive promoter. The reaction of aspartate production from fumaric acid and ammonium ion is catalyzed by aspartase(AspA) encoded by the gene ''aspA''<html><sup class="ref">[2]</sup></html>. (Figure. 1)
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Aspartate concentration gradient produced by guiders is gradually lost by diffusion as the time elapses. Therefore, in order to maintain the high worker bacteria density achieved by aspartate-dependent chemotaxis, it is required for the worker to be 'arrested' in the target location.
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To achive this, we have developed a mechanism that make bacteria less motile when cells are in target location by manipulating flagellum movement when they sense the substrate.
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Bacterial chemotaxis and flagellar movement has been extensively investigated and the molecular basis for E.coli chemotaxis has already been elucidated.
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:L-Asp is overproduced in the presence of enough AspA and the two substrates.
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According to previous study<html><sup class="ref">[3]</sup></html> and our experimental result, a gene named cheZ, which codes for protein involved in chemotactic signaling pathway, regulates cell motility.
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More specifically, cheZ gene in E.coli genomic DNA is constitutively expressed in physiological condition, causing the cell to become motile by affecting a flagellar motor binding protein called CheY.
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Therefore, cells are motile in the presence of cheZ and immotile in the absence of cheZ.
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(Learn more about chemotaxis pathway in E.coli)
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In order to make "worker" bacteria stay at the target site, we need to lower the cell motility in response to the substrate.
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:Note that because asp chemoattraction is native to ''E.coli'', guiders are also attracted to the substrate. Attracted guiders also produce aspartate, giving rise to a positive feedback in which guiders are continuously added and create even higher aspartate concentrations.
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Put in another way, we want the expression of cheZ to be activated in the absense of substrate and repressed in the presence of substrate.
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To achieve this, we utilize cheZ knockout strain of E.coli to eliminate baseline expression and devised the gene construct shown below, in which cheZ expression is repressed in a substrate-dependent manner (fig. 2).
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=Section 2: Substrate-induced Cell Arrest System=
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:As the reaction catalyzed by AspA is reversible, when the aspartate concentration around guiders becomes too high, the reaction reaches equilibrium and so additional aspartate cannot be produced. In addition, the substrates for AspA (fumaric acid and ammonium ion) may be limited. For these reasons, aspartate secretion by guiders does not last forever. The aspartate concentration gradient produced by guiders is gradually lost by diffusion as time elapses. Consequently, in order to maintain a high worker bacteria density, we attempted to "arrest" the bacteria at the target location to prevent bacteria from escaping. To achive this, we have developed a mechanism that make bacteria less motile when they are at the target location by manipulating flagellar movement.
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cIp -rbs - CheZ - d.Ter - Pi - rbs - cI - d.Ter
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:Bacterial chemotaxis and flagellar movement has been extensively investigated and the molecular basis for ''E.coli'' chemotaxis is understood. According to a previous study<html><sup class="ref">[3]</sup></html>, a gene named ''cheZ'', which codes for a protein involved in chemotactic signaling pathway, regulates cell motility. More specifically, ''cheZ'', constitutively expressed in ''E.coli'', phosphorylates a flagellar motor binding protein called CheY, making the cell motile. Therefore, cells are motile in the presence of ''cheZ'' and immotile in the absence of ''cheZ''.
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When the cell is outside of the target location, cI inhibitor is not expressed, whereas CheZ is expressed and the expression product rescues motility of CheZ- strain.
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:In order to make those working bacteria remain at a target site, we need to lower cell motility when they are exposed to the substrate. Put in another way, we want ''cheZ'' to be expressed in the absense of the substrate and repressed in the presence of the substrate.
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Once the cell gets inside the target location, substrate-responsive promoter is activated and cI inhibitor is expressed.
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cI inhibitor then inhibits the activity of cI promotor and thereby represses CheZ expression, which leads to substrate-induced loss of motility.
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:To achieve this, we utilize a ''cheZ'' knockout strain to eliminate baseline expression and devised the gene construct shown below, in which ''cheZ'' expression is repressed in a substrate-dependent manner.
 +
 
 +
:[[File:UT-Tokyo 36-3-29-5-SI-3-37-5.png|center|700px]]
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:When the cell does not detect the substrate, cI inhibitor is not expressed and therefore ''cheZ'' is expressed and the product rescues motility of the ''cheZ''- strain. Once the cell detects the substrate, the substrate-responsive promoter is activated and cI inhibitor is expressed. cI inhibitor then inhibits the activity of cI promotor and thereby represses ''cheZ'' expression, which leads to substrate-induced loss of motility.
 +
 
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:By combining these two mechanisms, cell density at the location of substrate is expected to be greatly elevated and maintained, so that enhanced working effectiveness will be achieved.
 +
 
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=Section3: UV Switch=
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:Our SMART System has a certain problem in that an initial induction is required for the activation of the System. So, our SMART system is at this point only applicable when transcription factors responding to the targeted substrate are already known. For instance, MerR and its responsive promoter can be combined with the SMART System for mercury remediation, since the transcriptional factors (MerR and responsive promoters) have already been investigated <html><sup class="ref">[4]</sup></html>.
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:To address this issue, we provided a well-characterized easy-inducible promoter as a genetic switch.
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:[[File:UT-Tokyo UV-switch.png|center|500px]]
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==References==
 
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<h2>References</h2>
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   <li id="ref_2">[2] Chao Y, Lo T, Luo N. Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli. Enzyme Microb. Technol. 2000;27(1-2):19-25.</li>
   <li id="ref_2">[2] Chao Y, Lo T, Luo N. Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli. Enzyme Microb. Technol. 2000;27(1-2):19-25.</li>
   <li id="ref_3">[3] Porter SL, Wadhams GH, Armitage JP. Signal processing in complex chemotaxis pathways. Nat. Rev. Microbiol. 2011;9(3):153-165.<li>
   <li id="ref_3">[3] Porter SL, Wadhams GH, Armitage JP. Signal processing in complex chemotaxis pathways. Nat. Rev. Microbiol. 2011;9(3):153-165.<li>
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  <li id="ref_4">[4] O'Halloran, T. V., Frantz, B., Shin, M. K., Ralston, D. M., & Wright, J. G. (1989). The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell, 56(1), 119-129.</li>
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Latest revision as of 01:50, 6 October 2011

System

Project Overview

As we stated in the Background page, our SMART E.coli project aims at improving the working efficiency of bioremediation. To achieve this goal, we have designed the following two sub-systems. We first developed a Cell Assembling System, in which E. coli "guiders" detect a substrate and attract other cells to themselves via chemoattraction, therefore elevating the bacterial density at the substrate location. We constructed and tested an inducible chemoattractant (L-aspartate) producing device using an enzyme aspA. Second, we devised a Cell Arrest System to prevent a time-dependent cell diffusion so that the elevated cell density is maintained. In this system, E. coli flagellum-regulating gene, cheZ, was utilized to realize E.coli "workers" anchoring at the site of substrate. By combining these two mechanisms, cell density at the location of substrate is expected to be greatly elevated and maintained, so that enhanced working effectiveness will be achieved.
To further reinforce the applicability of our SMART System, we also provided a "UV Switch", which enables us to switch on the SMART System even when a proper substrate-induced promoter does not exist. We investigated several UV-induced promoters, and successfully confirmed and characterized the UV-induced alteration of sulAp expression. Combining the UV Switch with our SMART System, it becomes possible to "switch on" the self-assembling and localization at the location of interest, which greatly enhances the future applications of this SMART System.

Section1: Substrate-induced Cell Assembling System

E.coli have a native property called 'chemotaxis,' and are attracted to certain substances called 'chemoattractants.' E.coli detect and move towards a higher concentration of the substances.
In our system we utilize this property to increase cell density. Specifically, we aim to realize substrate-responsive chemoattractant overproduction and a resultant cell assembly to the substrate.
Figure. 1 Aspartate production catalyzed by AspA
Figure. 1 Aspartate production catalyzed by AspA

The chemoattractant we have utilized is aspartate (L-Asp). Aspartate has previously been shown to act as a chemoattractant[1], which we confirmed by our own experiment, the cell diffusion assay. By making E.coli discharge aspartate, an aspartate concentration gradient around these bacteria is generated. Workers are attracted, creating a higher cell density around the aspartate-secreting E.coli.
We have devised a substrate-responsive aspartate overproduction mechanism. By utilizing this mechanism, guiders overproduce and secrete aspartate in response to a particular substrate and attract other bacteria to enhance cell density around the substrate.
The following is the gene construct we designed.
UT Tokyo 20-3-27-5.png
The coding sequence of AspA is inserted downstream of a substrate-responsive promoter. The reaction of aspartate production from fumaric acid and ammonium ion is catalyzed by aspartase(AspA) encoded by the gene aspA[2]. (Figure. 1)
L-Asp is overproduced in the presence of enough AspA and the two substrates.
Note that because asp chemoattraction is native to E.coli, guiders are also attracted to the substrate. Attracted guiders also produce aspartate, giving rise to a positive feedback in which guiders are continuously added and create even higher aspartate concentrations.

Section 2: Substrate-induced Cell Arrest System

As the reaction catalyzed by AspA is reversible, when the aspartate concentration around guiders becomes too high, the reaction reaches equilibrium and so additional aspartate cannot be produced. In addition, the substrates for AspA (fumaric acid and ammonium ion) may be limited. For these reasons, aspartate secretion by guiders does not last forever. The aspartate concentration gradient produced by guiders is gradually lost by diffusion as time elapses. Consequently, in order to maintain a high worker bacteria density, we attempted to "arrest" the bacteria at the target location to prevent bacteria from escaping. To achive this, we have developed a mechanism that make bacteria less motile when they are at the target location by manipulating flagellar movement.
Bacterial chemotaxis and flagellar movement has been extensively investigated and the molecular basis for E.coli chemotaxis is understood. According to a previous study[3], a gene named cheZ, which codes for a protein involved in chemotactic signaling pathway, regulates cell motility. More specifically, cheZ, constitutively expressed in E.coli, phosphorylates a flagellar motor binding protein called CheY, making the cell motile. Therefore, cells are motile in the presence of cheZ and immotile in the absence of cheZ.
In order to make those working bacteria remain at a target site, we need to lower cell motility when they are exposed to the substrate. Put in another way, we want cheZ to be expressed in the absense of the substrate and repressed in the presence of the substrate.
To achieve this, we utilize a cheZ knockout strain to eliminate baseline expression and devised the gene construct shown below, in which cheZ expression is repressed in a substrate-dependent manner.
UT-Tokyo 36-3-29-5-SI-3-37-5.png
When the cell does not detect the substrate, cI inhibitor is not expressed and therefore cheZ is expressed and the product rescues motility of the cheZ- strain. Once the cell detects the substrate, the substrate-responsive promoter is activated and cI inhibitor is expressed. cI inhibitor then inhibits the activity of cI promotor and thereby represses cheZ expression, which leads to substrate-induced loss of motility.
By combining these two mechanisms, cell density at the location of substrate is expected to be greatly elevated and maintained, so that enhanced working effectiveness will be achieved.

Section3: UV Switch

Our SMART System has a certain problem in that an initial induction is required for the activation of the System. So, our SMART system is at this point only applicable when transcription factors responding to the targeted substrate are already known. For instance, MerR and its responsive promoter can be combined with the SMART System for mercury remediation, since the transcriptional factors (MerR and responsive promoters) have already been investigated [4].
To address this issue, we provided a well-characterized easy-inducible promoter as a genetic switch.
UT-Tokyo UV-switch.png

References

  • [1] Topp S, Gallivan JP. Guiding bacteria with small molecules and RNA. J. Am. Chem. Soc. 2007;129(21):6807-6811.
  • [2] Chao Y, Lo T, Luo N. Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli. Enzyme Microb. Technol. 2000;27(1-2):19-25.
  • [3] Porter SL, Wadhams GH, Armitage JP. Signal processing in complex chemotaxis pathways. Nat. Rev. Microbiol. 2011;9(3):153-165.
  • [4] O'Halloran, T. V., Frantz, B., Shin, M. K., Ralston, D. M., & Wright, J. G. (1989). The MerR heavy metal receptor mediates positive activation in a topologically novel transcription complex. Cell, 56(1), 119-129.

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