Latest revision as of 16:04, 14 October 2009

Speed Control - Design

E. coli or Salmonella can swim around by the flagella rotating. When the flagella rotate counterclockwise, the bacteria form the forward motion which is called swimming. However, when the rotation is changed into clockwise, the bacteria tumble in place and are unable to swim (Fig 1).

Fig. 1 Genetic circuit related to cell movement iHKU

To control the speed of our Bactomotor, we aim at the direct swimming of bacteria for propelling the motor and the adjustable speed of swimming within a certain range. The aim is achieved by regulation of the expression level of cheZ gene. cheZ plays the key role here is due to its influence on the expression of cheY. A high level of phosphorylation of cheY protein in E. coli or Salmonellaleads to the majority of bacteria tumbling movement, while a low level of phosphorylation of cheY protein in E. coli or Salmonella is found in non-tumbling bacteria and cheZ can function to reduce the level of phophorylated cheY in the bacteria. Therefore, when increasing the expression level of CheZ gene, we can reduce the tumbling movement, which in turn can increase the swimming speed of the bacteria to achieve the manipulation of speed.

**Step 1--CheZ knockout**

By using lamda red system, recombineering could be applied to knock out the CheZ gene in the chromosome of E. coli or Salmonella. Homologous arma (about 50bp) were designed inside the CheZ gene and after recombination, the CheZ was replaced and destroyed by chloramphenicol resistance gene .

**Step 2--Controllable cheZ expression**

An inducible CheZ plasmid is tranformed into CheZ knockout strains. Therefore, by controlling cheZ expression level, we can implement the adjustable control over the speed of the bacteria and hence the motor.

There are two designs for cheZ plasmid.

First Design

The first design is to use lacI as a repressor to prevent any leaky expression in the absence of the inducer (IPTG or arabinose). When the bacteria were treated with IPTG or arabinose(switch on), the cheZ expression level could be regulated according to its concentration and hence the swimming speed of the bacteria.

Second Design

The second design is to use tetR as a repressor and ptet as the regulator, which can be induced by aTc. We suppose that by changing the concentration of aTc, the expression amount of protein cheZ will be altered, which results in the speed up and slow down of the swimmng.

HKU-HKBU speed control design second.png

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 =Speed Control - Design=
-''E. coli'' can swim around by the flagella rotating. To move forward, the flagella rotate counterclockwise and form the motion called swimming.  However, when the rotation changes to clockwise, the bacteria tumble in place and are unable to move.
+''E. coli'' or ''Salmonella'' can swim around by the flagella rotating. When the flagella rotate counterclockwise, the bacteria form the forward motion which is called swimming. However, when the rotation is changed into clockwise, the bacteria tumble in place and are unable to swim (Fig 1).
-[[Image:HKU-HKBU_speed_control_1.png | center]]
+[[Image:HKU-HKBU_speed_control_1.png | frame | center | Fig. 1 Genetic circuit related to cell movement [https://2008.igem.org/Team:iHKU/modeling iHKU]]]
-In our bacteria-motor, we aim at the direct swimming of bacteria for propelling the motor and the adjustable speed of swimming within a certain range. The aim is achieved mainly by regulation of the expression level of CheZ gene. CheZ plays the key role here is due to its influence on the expression of CheY. A high level of phosphorylation of CheY protein in ''E. coli'' leads to the majority of bacteria tumbling movement, while a low level of phosphorylation of CheY protein in ''E. coli'' is found in non-tumbling bacteria and CheZ can function to reduce the level of phophorylated CheY in the bacteria. Therefore, when increasing the expression level of CheZ gene, we can reduce the tumbling movement, which in turn can increase the swimming speed of the bacteria to achieve the manipulation of speed.
+To control the speed of our Bactomotor, we aim at the direct swimming of bacteria for propelling the motor and the adjustable speed of swimming within a certain range. The aim is achieved by regulation of the expression level of cheZ gene. cheZ plays the key role here is due to its influence on the expression of cheY. A high level of phosphorylation of cheY protein in ''E. coli'' or ''Salmonella''leads to the majority of bacteria tumbling movement, while a low level of phosphorylation of cheY protein in ''E. coli'' or ''Salmonella'' is found in non-tumbling bacteria and cheZ can function to reduce the level of phophorylated cheY in the bacteria. Therefore, when increasing the expression level of CheZ gene, we can reduce the tumbling movement, which in turn can increase the swimming speed of the bacteria to achieve the manipulation of speed.
-This speed control system is constructed by knocking out the CheZ gene and transform an inducible plasmid with CheZ for the implement of adjustable control over the speed of the bacteria and hence the motor. There are two designs for this plasmid.
+=='''Step 1--''CheZ'' knockout'''==
+By using lamda red system, recombineering could be applied to knock out the ''CheZ'' gene in the chromosome of ''E. coli'' or ''Salmonella''. Homologous arma (about 50bp) were designed inside the ''CheZ'' gene and after recombination, the ''CheZ'' was replaced and destroyed by chloramphenicol resistance gene .
+=='''Step 2--Controllable ''cheZ'' expression'''==
+An inducible CheZ plasmid is tranformed into ''CheZ'' knockout strains. Therefore, by controlling cheZ expression level, we can implement the adjustable control over the speed of the bacteria and hence the motor.
+There are two designs for ''cheZ'' plasmid.
 ===First Design===
-The first design is to use lacI as a repressor to prevent any leaky expression in the absence of the inducer. The promoter in this construct is Plac, which can be induced by IPTG.
+The first design is to use '''lacI''' as a repressor to prevent any leaky expression in the absence of the inducer (IPTG or arabinose). When the bacteria were treated with IPTG or arabinose(switch on), the'' cheZ'' expression level could be regulated according to its concentration and hence the swimming speed of the bacteria.
 [[Image:HKU-HKBU_speed_control_design_first.png|center]]
-In the proposed model, by changing the concentration of IPTG, we would be able to change the level of expression of CheZ and therefore the swimming speed of the bacteria.
 ===Second Design===
-The second design is to use tetR as a repressor and Ptet as the regulator, which can be induced by aTc.
+The second design is to use '''tetR''' as a repressor and '''ptet''' as the regulator, which can be induced by aTc. We suppose that by changing the concentration of aTc, the expression amount of protein cheZ will be altered, which results in the speed up and slow down of the swimmng.
 [[Image:HKU-HKBU_speed_control_design_second.png|center]]
-We suppose that by changing the concentration of aTc, the expression amount of protein CheZ will be changed, which results in the alternation of the swimming speed.
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Team:HKU-HKBU/speed control design

From 2009.igem.org