|
|
| Line 84: |
Line 84: |
| | <tr> | | <tr> |
| | <td class="bodyText"><p align="justify"> | | <td class="bodyText"><p align="justify"> |
| - | The idea of our project is to make the system work akin to this: <br /><br /> | + | <strong>Singing Bacteria!</strong><br><br> |
| - | RcnR works as a natural repressor for rncA. With nickel present, RcnR will change its conformation by binding to it, and consequently, the complex will stop the repression of the transcription of rcnA, which is the metal efflux pump protein that controls the nickel concentration in the interior of the cell. The trigger (input) to create this modification will be given by adding AHL to the MEDIA. The ALH will bind to LuxR and this complex will induce the cI transcription (cI is the repressor for the modified lambda phage with an extra LVA tail for its swift degradation). Since cl is activated, it will repress the rcnA transcription, restricting the nickel exclusion and increasing its concentration within the cell. The signal will be eliminated promptly due to the constant presence of aiiA degradating AHL and the fast degradation of cI, therefore the system will return to its initial state.
| + | The overall idea in our project for the 2008 iGEM competition is to make bacteria sing. <strong>And how can we possibly achieve that?</strong><br><br> |
| | + | The main idea behind this is to control E. coli’s nickel efflux pump (RcnA). This way we’ll be able to predict the extracellular nickel concentration at any time in function of the controling signal (AHL). Electrodes will be sensing the electrical conductivity in the extracellular medium and sending that information to a computer. The computer will interpret this information and emit a sound depending on the nickel concentration at that time. This way, bacteria are “singing”.<br><br> |
| | + | The proteins and compounds involved in our model are the following:<br> |
| | + | <li><b>RcnA.</b> This is E. coli’s nickel efflux pump. We’re using a RcnA-mutant E. coli strain with a plasmid with RcnA under the control of cI* and RcnR (both repressors).<br> |
| | + | <li><b>RcnR.</b> The natural repressor of RcnA. When there’s nickel present, RcnR changes its conformation and stops repressing RcnA. <br> |
| | + | <li><b>cI*.</b> The λ phage repressor. The transcription of cI* is under the control of the activator dimer AHL:LuxR. cI is modified with a LVA tail so it is quickly degraded (hence the *).<br> |
| | + | <li><b>LuxR.</b> Used here as an activator of cI*. It is under a constitutive promoter (pTetR).<br> |
| | + | <li><b>AHL.</b> This is the input signal. AHL forms a dimer with LuxR and starts the transcription of cI*. <br> |
| | + | <li><b>AiiA.</b> This protein mediates the degradation of AHL. It is placed under a weak promoter (modified pLacZ).<br><br> |
| | + | Here is a simplified diagram of the whole process: |
| | </p> | | </p> |
| | + | <img src="https://static.igem.org/mediawiki/2008/b/b9/Our_Project_01.jpg" width="580"> |
| | + | <p align="justify">So, here is the formal definition:<br> |
| | + | The objective of this project is to modulate the extracellular nickel concentration through the regulation of its efflux pump. We plan to measure the change in the nickel concentration, and this can be achieved by measuring the change in the conductivity of the medium. These data will be interpreted and converted into a sound depending on the concentration read.<br><br> |
| | + | Our system is conformed by two regulation mechanisms. The first of these is the one controlled by us through AHL. AHL enters the cell and forms a dimer with LuxR, which is under a constitutive promoter. This dimer serves as an activator of cI*, which represses RcnA. In this way we can express the concentration (and therefore the activity) of RcnA as a function of AHL. The second of these mechanisms is the natural regulation of the pump (RcnA) in response to the intracellular nickel concentration. When there is no nickel inside the cell, RcnR represses RcnA. However, when nickel enters the cell, it forms a dimer with RcnR and changes its conformation so it no longer represses RcnA. RcnA is then free to start pumping Ni out of the cell. We are keeping this because it is damaging to the bacteria to have the pump always on, and otherwise it would need a constant supply of AHL.<br><br> |
| | + | The final result will be a biological system capable of modifying its surrounding medium. In addition, our measurements will allow us to model the dynamic behavior of the pump and the intra and extracellular nickel concentrations.<br> |
| | + | ----------- ------------------------------------- --------------- --------------------------------- --------------------------- </p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
| Line 93: |
Line 108: |
| | </tr> | | </tr> |
| | <tr> | | <tr> |
| - | <td class="bodyText"><p align="justify">--- - - --- -- -- -- -- -- -- -- - ------ -----. --- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ---- --- --- -- - ------ -- ---- --- --- --- -- -- ----- ---, --- - - --- -- -- -- -- -- -- -- - ------ -----. | + | <td class="bodyText"><p align="justify"> |
| - | --- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ----. </p>
| + | ----------- ------------------------------------- --------------- --------------------------------- --------------------------- </p> |
| | </td> | | </td> |
| | </tr> | | </tr> |
 |
LCG-UNAM-Mexico |
 |
| iGEM 2008 TEAM |
 |
 |
| |
|
|
|
|
| Our Project |
Singing Bacteria!
The overall idea in our project for the 2008 iGEM competition is to make bacteria sing. And how can we possibly achieve that?
The main idea behind this is to control E. coli’s nickel efflux pump (RcnA). This way we’ll be able to predict the extracellular nickel concentration at any time in function of the controling signal (AHL). Electrodes will be sensing the electrical conductivity in the extracellular medium and sending that information to a computer. The computer will interpret this information and emit a sound depending on the nickel concentration at that time. This way, bacteria are “singing”.
The proteins and compounds involved in our model are the following:
RcnA. This is E. coli’s nickel efflux pump. We’re using a RcnA-mutant E. coli strain with a plasmid with RcnA under the control of cI* and RcnR (both repressors).
RcnR. The natural repressor of RcnA. When there’s nickel present, RcnR changes its conformation and stops repressing RcnA.
cI*. The λ phage repressor. The transcription of cI* is under the control of the activator dimer AHL:LuxR. cI is modified with a LVA tail so it is quickly degraded (hence the *).
LuxR. Used here as an activator of cI*. It is under a constitutive promoter (pTetR).
AHL. This is the input signal. AHL forms a dimer with LuxR and starts the transcription of cI*.
AiiA. This protein mediates the degradation of AHL. It is placed under a weak promoter (modified pLacZ).
Here is a simplified diagram of the whole process:
So, here is the formal definition:
The objective of this project is to modulate the extracellular nickel concentration through the regulation of its efflux pump. We plan to measure the change in the nickel concentration, and this can be achieved by measuring the change in the conductivity of the medium. These data will be interpreted and converted into a sound depending on the concentration read.
Our system is conformed by two regulation mechanisms. The first of these is the one controlled by us through AHL. AHL enters the cell and forms a dimer with LuxR, which is under a constitutive promoter. This dimer serves as an activator of cI*, which represses RcnA. In this way we can express the concentration (and therefore the activity) of RcnA as a function of AHL. The second of these mechanisms is the natural regulation of the pump (RcnA) in response to the intracellular nickel concentration. When there is no nickel inside the cell, RcnR represses RcnA. However, when nickel enters the cell, it forms a dimer with RcnR and changes its conformation so it no longer represses RcnA. RcnA is then free to start pumping Ni out of the cell. We are keeping this because it is damaging to the bacteria to have the pump always on, and otherwise it would need a constant supply of AHL.
The final result will be a biological system capable of modifying its surrounding medium. In addition, our measurements will allow us to model the dynamic behavior of the pump and the intra and extracellular nickel concentrations.
----------- ------------------------------------- --------------- --------------------------------- ---------------------------
|
| Project Details |
----------- ------------------------------------- --------------- --------------------------------- ---------------------------
|
--- - - --- -- -- -- -- -- -- -- - ------ -----. --- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ---- --- --- -- - ------ -- ---- --- --- --- -- -- ----- ---, --- - - --- -- -- -- -- -- -- -- - ------ -----.
--- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ----.
|
--- - - --- -- -- -- -- -- -- -- - ------ -----. --- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ---- --- --- -- - ------ -- ---- --- --- --- -- -- ----- ---, --- - - --- -- -- -- -- -- -- -- - ------ -----.
--- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ----.
|
--- - - --- -- -- -- -- -- -- -- - ------ -----. --- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ---- --- --- -- - ------ -- ---- --- --- --- -- -- ----- ---, --- - - --- -- -- -- -- -- -- -- - ------ -----.
--- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ----.
|
| Results |
--- - - --- -- -- -- -- -- -- -- - ------ -----. --- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ---- --- --- -- - ------ -- ---- --- --- --- -- -- ----- ---, --- - - --- -- -- -- -- -- -- -- - ------ -----.
--- ------ --- ---- -- --- ----- -- -- -- ------ ---- ---- - -- - ---- - ----- ---- ---, - ----- --- ----- ---- ---- ---- -- --- --- --- --- -- ---- ---------- - ------ ---- --- ---- ----- --- --- ----- ---- ----- ------ ---- -- -- --- ------- - ----- --- ----.
|
| |
| |
|
|
|
|
|
|
"
