Team:WITS-CSIR SA/Project/Concept

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<p>The WITS_CSIR_SA iGEM 2011 team decided to create a microscopic biological communication network, in which there will be the transfer of data between bacterial populations as chemical signals. This type of system has been dubbed “Biotweet”. The generic framework of “Biotweet” may serve as the basis for the functioning of complex biological communication networks and may be useful in various applications, including those of the medical and industrial sectors.</p>
<p>The WITS_CSIR_SA iGEM 2011 team decided to create a microscopic biological communication network, in which there will be the transfer of data between bacterial populations as chemical signals. This type of system has been dubbed “Biotweet”. The generic framework of “Biotweet” may serve as the basis for the functioning of complex biological communication networks and may be useful in various applications, including those of the medical and industrial sectors.</p>
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<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Biotweet: A framework for bacterial networks using programmed bacterial motility '">
<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Biotweet: A framework for bacterial networks using programmed bacterial motility '">
Any network requires the establishment of connections between nodes of the network, allowing for the transport of node-to-node data packets. With “Biotweet”, we have attemptedto construct such a network within a biological network: a challenging task, considering that bacterial cells cannot be linked by wires. Analogous to the transfer of data as electronic packets in a computer network, the concept of engineering bacteria to transport packets of chemical signals between bacterial populations arose. In this way, the bacteria themselves can form the network connections. In order to do this, we decided to reprogram the motility of bacteria, such that each bacterium travels in a stimulus-directed fashion (eg. chemotactic response) to specific points of the network (the nodes), where they can send and receive signals. Such a stimulus should be unique to the bacterium and not be part of the natural environment. With these specifications in mind, we needed a way of manipulating the chemotactic behaviour of bacteria that would allow for us to easily adapt the bacterial chemotactic response to the stimuli associated with any application. Specifically, the chemotactic response in E.coli lends itself to easy manipulation. The activation of CheZ allows for counter-clockwise (CCW) flagellar activity resulting in a “run and tumble” movement. By linking CheZ expression to post-transcriptional activation by a specific anayte (using for example a analyte-specific riboswitch), bacterial motility can be guided and controlled (Fig.1). For more on bacterial chemotaxis,<a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Motility">See here</a> </p>
Any network requires the establishment of connections between nodes of the network, allowing for the transport of node-to-node data packets. With “Biotweet”, we have attemptedto construct such a network within a biological network: a challenging task, considering that bacterial cells cannot be linked by wires. Analogous to the transfer of data as electronic packets in a computer network, the concept of engineering bacteria to transport packets of chemical signals between bacterial populations arose. In this way, the bacteria themselves can form the network connections. In order to do this, we decided to reprogram the motility of bacteria, such that each bacterium travels in a stimulus-directed fashion (eg. chemotactic response) to specific points of the network (the nodes), where they can send and receive signals. Such a stimulus should be unique to the bacterium and not be part of the natural environment. With these specifications in mind, we needed a way of manipulating the chemotactic behaviour of bacteria that would allow for us to easily adapt the bacterial chemotactic response to the stimuli associated with any application. Specifically, the chemotactic response in E.coli lends itself to easy manipulation. The activation of CheZ allows for counter-clockwise (CCW) flagellar activity resulting in a “run and tumble” movement. By linking CheZ expression to post-transcriptional activation by a specific anayte (using for example a analyte-specific riboswitch), bacterial motility can be guided and controlled (Fig.1). For more on bacterial chemotaxis,<a href="https://2011.igem.org/Team:WITS-CSIR_SA/Project/Motility">See here</a> </p>
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<p>Fig 2.Using ligand-responsive riboswitch activation of CheZ expression. CheZ can be fused to a reporter and direct bacterial motility in a ligand-dependent manner up a concentration gradient. </p>
<p>Fig 2.Using ligand-responsive riboswitch activation of CheZ expression. CheZ can be fused to a reporter and direct bacterial motility in a ligand-dependent manner up a concentration gradient. </p>
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<div data-dojo-type="dijit.TitlePane" data-dojo-props="title:'Designing a “send” and “receive” network using riboswitch-controlled bacterial motility'">
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<p>The aim was to reprogram E. coli (which lack natural CheZ functionality) with riboswitch-activated CheZ. This enables bacteria to search, localise to the riboswitch activator, and return to a set location in order to report. This, in principle, will show that bacteria can be programmed to move in a directed manner towards a new stimulus and establish a connection between two points in space. The generic design for this is depicted below. The “send” module is essentially a riboswitch-activated CheZresponsive to analyte A, and the “receive” module is a riboswitch-activated CheZ responsive to analyte B. We have decided to include two fluorescent reporters as fusion proteins since both N- and C-terminal CheZ fusions are well-tolerated (Gallivan, 2007). To ensure that the “send” and “receive” modules act independently, Analyte B can be in a biphasic toggle switch with repressor Z. Therefore Z, which represses Cre-recombinase, is activated flipping the toggle (by a critical concentration of AnalyteA). Cre recombination allows for irreversible switching of constitutive promoter activity (by lox site recombination) and convert motility from “send” to “receive” such that bacterial motility is now dictated by a concentration gradient of analyte B.</p>
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<p>Fig 3.“send” and “receive” riboswitch-controlled motility module. Featured is also an Analyte B-responsive toggle switch which activates Crerecombinase expression. Cre, in turn, causes “receive” motility activationby flipping constitutively active promoter, and allowing motility to be controlled by Analyte A.</p>
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<p>Unfortunately, other than Tetracycline (Dox) riboswitches for use in yeast, at present, there isn’t an engineeredriboswitch which is sensitive to a known transcriptional repressor. Therefore, we designed system which allows the “receive” function  (Crerecombinase) to be activated by IPTG (lacI). Also, we focused this design on the use of two known synthetic riboswitches – against theophylline and atrazine (see riboswitch design considerations below).</p>
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Revision as of 23:01, 19 September 2011

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd"> Biotweet - Concept

Biotweet iGem
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The WITS_CSIR_SA iGEM 2011 team decided to create a microscopic biological communication network, in which there will be the transfer of data between bacterial populations as chemical signals. This type of system has been dubbed “Biotweet”. The generic framework of “Biotweet” may serve as the basis for the functioning of complex biological communication networks and may be useful in various applications, including those of the medical and industrial sectors.


Any network requires the establishment of connections between nodes of the network, allowing for the transport of node-to-node data packets. With “Biotweet”, we have attemptedto construct such a network within a biological network: a challenging task, considering that bacterial cells cannot be linked by wires. Analogous to the transfer of data as electronic packets in a computer network, the concept of engineering bacteria to transport packets of chemical signals between bacterial populations arose. In this way, the bacteria themselves can form the network connections. In order to do this, we decided to reprogram the motility of bacteria, such that each bacterium travels in a stimulus-directed fashion (eg. chemotactic response) to specific points of the network (the nodes), where they can send and receive signals. Such a stimulus should be unique to the bacterium and not be part of the natural environment. With these specifications in mind, we needed a way of manipulating the chemotactic behaviour of bacteria that would allow for us to easily adapt the bacterial chemotactic response to the stimuli associated with any application. Specifically, the chemotactic response in E.coli lends itself to easy manipulation. The activation of CheZ allows for counter-clockwise (CCW) flagellar activity resulting in a “run and tumble” movement. By linking CheZ expression to post-transcriptional activation by a specific anayte (using for example a analyte-specific riboswitch), bacterial motility can be guided and controlled (Fig.1). For more on bacterial chemotaxis,See here

Fig 1.The reprogramming of bacterial chemotactic response by using post-transcriptional activation of CheZ using a analyte-responsive riboswitch. CheZ expression results in counter-clockwise flagellar rotation and a “run and tumble” movement.


Firstly, we have chosen to use artificial riboswitches to control the expression of the flagella rotation regulator protein CheZ. To do this, we borrowed heavily from studies which have described riboswitch-controlled bacterial chemotaxis(Topps and Gallivan. 2007).Riboswitches are ligand-inducible RNA protein expression regulators that are comprised of an aptamer domain and an expression platform (Gallivan, 2007). The aptamer is a sequence of nucleotides that is designed to specifically bind to ligands, while the expression platform consists of a ribosome binding site (RBS) and a downstream coding region (CDS). When the specific ligand or analyte binds to the aptamer domain, the riboswitch undergoes a structural change. This results in the exposure of the RBS and the expression of the downstream CDS (Fig.2). Using this riboswitch mechanism, the expression of CheZ can be regulated in a ligand concentration-dependent manner, outside the control of the natural chemotaxis pathway (Fig2, bottom panel and right). Topps and Gallivan (2007) demonstrated that these riboswitches can be used to reprogram the chemotactic response of bacteria, so that they move up a concentration gradient, towards the source of the stimulus that activates the riboswitch (a process known aspseudotaxis). Bacteria can theoretically be reprogrammed to respond to any stimulus, if the appropriate riboswitch is made. This provides the versatility needed for biological network connections to be established in any application. Furthermore, this eliminates the need to engineer novel chemoreceptors.

Fig 2.Using ligand-responsive riboswitch activation of CheZ expression. CheZ can be fused to a reporter and direct bacterial motility in a ligand-dependent manner up a concentration gradient.


The aim was to reprogram E. coli (which lack natural CheZ functionality) with riboswitch-activated CheZ. This enables bacteria to search, localise to the riboswitch activator, and return to a set location in order to report. This, in principle, will show that bacteria can be programmed to move in a directed manner towards a new stimulus and establish a connection between two points in space. The generic design for this is depicted below. The “send” module is essentially a riboswitch-activated CheZresponsive to analyte A, and the “receive” module is a riboswitch-activated CheZ responsive to analyte B. We have decided to include two fluorescent reporters as fusion proteins since both N- and C-terminal CheZ fusions are well-tolerated (Gallivan, 2007). To ensure that the “send” and “receive” modules act independently, Analyte B can be in a biphasic toggle switch with repressor Z. Therefore Z, which represses Cre-recombinase, is activated flipping the toggle (by a critical concentration of AnalyteA). Cre recombination allows for irreversible switching of constitutive promoter activity (by lox site recombination) and convert motility from “send” to “receive” such that bacterial motility is now dictated by a concentration gradient of analyte B.

Fig 3.“send” and “receive” riboswitch-controlled motility module. Featured is also an Analyte B-responsive toggle switch which activates Crerecombinase expression. Cre, in turn, causes “receive” motility activationby flipping constitutively active promoter, and allowing motility to be controlled by Analyte A.

Unfortunately, other than Tetracycline (Dox) riboswitches for use in yeast, at present, there isn’t an engineeredriboswitch which is sensitive to a known transcriptional repressor. Therefore, we designed system which allows the “receive” function (Crerecombinase) to be activated by IPTG (lacI). Also, we focused this design on the use of two known synthetic riboswitches – against theophylline and atrazine (see riboswitch design considerations below).

Gallivan JP. Toward reprogramming bacteria with small molecules and RNA. Curr Opin Chem Biol 2007;11:612-9

Topp S, Gallivan JP. Guiding bacteria with small molecules and RNA. J Am Chem Soc 2007;129:6807-11.

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