Team:Hong Kong HKUST/riboregulator/CR TA Feature Page

From 2014.igem.org

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<strong>Abstract: </strong>
<strong>Abstract: </strong>
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The project we selected was to design and build a biomechanical printer; composed of a two dimensional plotter  
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Calgary 2007 wanted to design and build a biomechanical printer. The printer composed of a two dimensional plotter equipped with a red laser. The plotter would receive instruction from a software that can translate computer images into instructions. E. coli would respond to the laser light. The red laser light would trigger the expression of beta-agarase enzyme which can degrade the agar polymer that cell rest on. This would produce “bacterial lithography”, where the E. coli exposed to red laser light would dissolved that agar and form a picture.</p>
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equipped with a red laser, software to translate computer images into instructions for the plotter, and E. coli  
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<p>For Calgary 2007’s project to work, they needed a precise control of the gene of interest. To have such control, they decided to use the riboregulator system to prevent accidental expression of gene of interest.</p>
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cells engineered to respond to the laser light. Bacteria are spread in a solid lawn on the plate,
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or mixed in the media before pouring the plate. The response triggered by this biological circuit will produce beta agarase,
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an enzyme which degrades the agar polymer that the cells rest on.
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<p>The printer can then be used to "draw" high resolution images on the bacteria with the laser. The bacteria will then dissolve the agar where the laser was shone. This results in Bacterial Lithography, where the dissolved agar forms a picture.</p>
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<p>We also chose a second project to include in our entry to the competition this year. That is an in Silico Biobrick Evolution system. The purpose of this project is to design a system that will accept user entered parameters and use them to search through the registry database. Using the given parameters the system will try to construct circuits (a series of biobricks) that will produce the desired  product. More information on this project can be found in our evoGEM sections.</p>
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Our project attacks the following problem: can one engineer viruses to selectively kill or modify specific subpopulations of target cells, based on their RNA or protein expression profiles?
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Caltech 2007 wanted to engineer viruses to selectively kill or modify specific subpopulation of target cells, based on their RNA or protein expression profiles. To prove the concept, they decided to use bacteriophage λ and E. coli. Caltech 2007 tried to engineer a bacteriophage λ to target and lyse specific subpopulations of E. coli. In their design, the developmental genes of the virus essential for virus infection and survival are regulated by cis-repressing RNA (lock). Only the target E.coli contain the trans-activating RNA (key) to activate the expression of the virus developmental genes. Once the genes are activated, the target cells will go through the lysis process. Meanwhile, non-target cells won’t be affected by the virus because the non-target cells lack the key to activate the expression of the virus developmental genes.
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This addresses an important issue in gene therapy, where viruses engineered for fine target discrimination would selectively kill only those cells over- or under-expressing specific disease or cancer associated genes. Alternatively, these viruses could be used to discriminate between strains in a bacterial co-culture, allowing strain-specific modification or lysis.
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This is clearly an ambitious goal, so we brainstormed a simple model of this problem suitable for undergraduates working over a summer. The bacteriophage λ is a classic, well studied virus capable of infecting E. coli, another classic model genetic system. We therefore seek to engineer a λ strain targeted to lyse specific subpopulations of E. coli based on their transcriptional profiles. Together, λ and E. coli provide a tractable genetic model for this larger problem, while hopefully providing lessons applicable to more ambitious, future projects.
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<h2><a href="https://2009.igem.org/Team:TUDelft">Delft 2009</a></h2>
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<h2><a href="https://2009.igem.org/Team:TUDelft">TU Delft 2009</a></h2>
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<h5 class= "page_link"><a href="https://2009.igem.org/Team:TUDelft/Lock/Key_library">Delft Riboregulator page </a></h5>
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<h5 class= "page_link"><a href="https://2009.igem.org/Team:TUDelft/Lock/Key_library">TU Delft Riboregulator page </a></h5>
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<strong>Abstract: </strong>
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Our project aims to design a genetic device able to count and memorize the occurrences of an input signal. We achieved this by utilization of auto-inducing loops, that act as memory units, and an engineered riboregulator, acting as an AND gate. The design of the device is modular, allowing free change of both input and output signals. Each increase of the counter results in a different output signal. The design allows implementation of any number of memory units, as the AND gate design enables to extend the system in a hassle-free way. In order to tweak bistable autoinducing loops we need a very fast and robust method for characterizing parts. For this we have created a genetic algorithm that will enable us to find parameters of the parts used in the design. It also allows the combination of data from multiple experiments across models with overlapping components
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TU Delft 2009 wanted to build an improved cell to cell communication system.  They wanted to construct an E.coli strain that can pass GFP signals through conjugation only once. When “initiator cells” which contains the “signal plasmid” for the communication system, is added to a cell culture, the initiator cells (donor cells) conjugate with other cells “non-initiator” cells in the culture. During the conjugation, the signal plasmid is transferred to the recipient cells. The signal plasmid encodes for an entry exclusion protein, an endonuclease, and GFP. It has a restriction site of the endonuclease. The entry exclusion protein prevents backward conjugation, the endonuclease and the restriction site can reset the cell back to off state, and finally GFP can a reporter to visualize where the signal is. The expression of endonuclease should be delayed because cells, which just received the signal plasmid, require time to conjugate and pass down the signal plasmid to the recipient cells. TU Delft 2009 tried to solve this problem by introducing a delay device.  The riboregulator system was used for their RNA-based delay system. By having another level of regulation between the reception of signal and expression of self-destructing protein (e.g. endonuclease), Delft 2009 believed that there could be delayed expression of the self-destructing protein. TU Delft 2009 also designed an algorithm called Ribo Key & Lock Generator that can generate sequences for lock and key riboregulators when RBS sequence is used as an input.
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Delft Riboregulator references   
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TU Delft Riboregulator references   
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In our project we aim to create a cell-to-cell communication system that allows the propagation of a multi-task message with the capability of being reset. To achieve this, the system will include a reengineered conjugation system, a time-delay genetic circuit and a self-destructive plasmid. This system could be the basis for creating a long distance biosensor and/or be applied in reducing antibiotic resistance of bacteria. Furthermore, we have done a parallel research on the different perceptions of iGEM participants and supervisors on ethical issues in synthetic biology. We focused on the consequences of the ultimate conditions of the top-down and bottom-up approaches as applied in biology.  
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“Our project aims to design a genetic device able to count and memorize the occurrences of an input signal. We achieved this by utilization of auto-inducing loops, that act as memory units, and an engineered riboregulator, acting as an AND gate. The design of the device is modular, allowing free change of both input and output signals. Each increase of the counter results in a different output signal. The design allows implementation of any number of memory units, as the AND gate design enables to extend the system in a hassle-free way. In order to tweak bistable autoinducing loops we need a very fast and robust method for characterizing parts. For this we have created a genetic algorithm that will enable us to find parameters of the parts used in the design. It also allows the combination of data from multiple experiments across models with overlapping components.
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<h2><a href="https://2008.igem.org/Team:KULeuven">K.U. Leuven 2008 </a></h2>
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<h5 class= "page_link"><a href="https://2008.igem.org/Team:KULeuven/Project/Reset">K.U Leuven 2008 Riboregulator page</a></h5>
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Imagine a bacterium that produces a drug when and where it is needed in the human body. It would have several advantages over classical drugs and could have many medical applications. In this framework we proudly present our team's project: Dr. Coli, the bacterial drug delivery system. Dr. Coli senses the disease signal and produces the appropriate amount of drugs to meet the individual patient's needs. And when the patient is cured, Dr. Coli self-destructs. To do this, a molecular timer registers the time since the last disease signal sensed. But when the disease flares up again, this timer is reset and drug production is resumed. Within the time frame of the iGEM competition, we developed a proof of concept of Dr. Coli. The most important assets are massive reuse of standard biobricks, different control mechanisms and extensive modeling.
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<h2><a href="https://2009.igem.org/Team:KULeuven">K.U. Leuven 2009 </a></h2>
<h2><a href="https://2009.igem.org/Team:KULeuven">K.U. Leuven 2009 </a></h2>

Revision as of 01:06, 18 October 2014



Riboregulator Feature Page

In this feature page, several teams that have worked with riboregulators are highlighted. More outstanding riboregulators are also identified according to the these criteria: availability from Part Registry, usage frequency, and availability of characterization information of the riboregulator system. Finally, abstracts or summary of different teams’ projects that used or planned to use riboregulators are included; links are provided to each team’s riboregulator page for more information. This feature page can help users to quickly scan through the available riboregulators, and determine whether the riboregulator system is feasible for the project in mind by comparing previous projects that have used the riboregulator system.

Introduction to riboregulators

Figure 1.

Artificial riboregulator system used to control post-transcriptional regulation

Regulatory RNAs are small RNA that regulates biological processes such as transcription or translation. The use of regulatory RNAs has been a great interest in the field of synthetic biology because it provides an additional level of regulation for biological circuits and systems. Regulatory RNAs have also used by many iGEM teams. We have identified 7 teams that have used cis-repressing (CR) and trans-activating (TA) riboregulator system and more teams that have used riboswitches. For example, UC Berkeley 2005, UC Berkeley 2006 and Caltech 2007 contributed many CR and TA devices to the Registry.

Artificial cis-repressing and trans-activating riboregulator system was introduced to the iGEM community by the UC Berkeley iGEM 2005 team. The riboregulator system as a whole acts to regulate translation at the RNA level. One component of the system ,crRNA, which contains a cis-repressing sequence 5’ of the RBS, RBS, and gene of interest. The cis-repressing sequence can form a loop form complementary base pairs with the RBS , blocking the ribosome entry to RBS. crRNA is also commonly described as a “lock” because it “locks” the RBS and prevent translation. The “key” to this system is the taRNA. It can interact (in trans) with the cis-repressing sequence to unlock the RBS and therefore to activate translation (Figure 1.). The benefits of this system, as described in Isaacs et al.’s paper, are leakage minimization, fast response time, tunability, independent regulation of multiple genes etc1.

The benefits of this system, as described in Isaacs et al.'s paper, are leakage minimization, fast response time, tunability, independent regulation of multiple genes etc1.








Distinguished teams worked with riboregulators

Table 1. Summary of some of the teams that made contributions to the iGEM community regarding riboregulators

Team Track Chassis Contribution
Berkeley 2005 This team has not been assigned to a track. N/A Introduced modified version of Isaacs’ crR12 and taR12 cognate pair to the Registry
Berkeley 2006 This team has not been assigned to a track. E. coli Modified locks and keys to improve riboregulator system function
Caltech 2007 Foundational Research E. coli Characterized some locks and keys
TU Delft 2009 Information Processing E. coli Designed an algorithm that can generate lock and key sequences

Riboregulator parts in the Registry

Please goto Catalog Page for more part related to riboregulator

Table 2. List of more outstanding riboregulators parts in the Part Registry

star Name Description Length Designed by Group
BBa_J01008 Riboregulator key 1 94 Golden Bear iGEM2005
BBa_J01010 Riboregulator Lock 1 40 Golden Bear iGEM2005
BBa_J23066 [key3c][key3d][B0015] iGEM2006_Berkeley 8 Sample in stock
BBa_J23078 lock3i iGEM2006_Berkeley 15 It’s complicated
BBa_J23008 key3c iGEM2006_Berkeley 13 Sample in stock

Abstracts or summary of different teams’ projects that worked with riboregulators

The usage of the riboregulator system can be versatile; Different teams utilized the system in different ways. In general, introducing the riboregulator system to a certain circuit allows another level regulation. The new level of regulation then can give possible outcomes such as: specific interaction/communication (UC Berkeley 2006/ Caltech 2007); minimization of leakage by introducing post-transcriptional repression (Calgary 2007); and modulation of different concentrations of desired outcome (K.U. Leuven 2009).

One drawback of riboregulator system in the context of iGEM is that teams that originally have riboregulator incorporated in their systems drop the use of riboregulator of because of time restraint (Calgary 2007). This is because when riboregulators are used to introduce another level of regulation, it becomes a supplementary part of the project; Usually, even without the extra level of regulation, the teams can prove the functionality of their systems. Therefore when the time is limited, and when the teams prioritize the things they need to do, they end up dropping the riboregulator system.

UC Berkeley 2006

Abstract: “Networks of interacting cells provide the basis for neural learning. We have developed the process of addressable conjugation for communication within a network of E. coli bacteria. Here, bacteria send messages to one another via conjugation of plasmid DNAs, but the message is only meaningful to cells with a matching address sequence. In this way, the Watson Crick base-pairing of addressing sequences replaces the spatial connectivity present in neural systems. To construct this system, we have adapted natural conjugation systems as the communication device. Information contained in the transferred plasmids is only accessible by "unlocking" the message using RNA based 'keys'. The resulting addressable conjugation process is being adapted to construct a network of NAND logic gates in bacterial cultures. Ultimately, this will allow us to develop networks of bacteria capable of trained learning.”

Berkeley Riboregulator Reference :

  1. Isaacs, Dwyer DJ, Ding C, Pervouchine DD, Cantor CR, Collins JJ. "Engineered riboregulators enable post-transcriptional control of gene expression"

Calgary 2007

Abstract: Calgary 2007 wanted to design and build a biomechanical printer. The printer composed of a two dimensional plotter equipped with a red laser. The plotter would receive instruction from a software that can translate computer images into instructions. E. coli would respond to the laser light. The red laser light would trigger the expression of beta-agarase enzyme which can degrade the agar polymer that cell rest on. This would produce “bacterial lithography”, where the E. coli exposed to red laser light would dissolved that agar and form a picture.

For Calgary 2007’s project to work, they needed a precise control of the gene of interest. To have such control, they decided to use the riboregulator system to prevent accidental expression of gene of interest.

Caltech 2007

Abstract: Caltech 2007 wanted to engineer viruses to selectively kill or modify specific subpopulation of target cells, based on their RNA or protein expression profiles. To prove the concept, they decided to use bacteriophage λ and E. coli. Caltech 2007 tried to engineer a bacteriophage λ to target and lyse specific subpopulations of E. coli. In their design, the developmental genes of the virus essential for virus infection and survival are regulated by cis-repressing RNA (lock). Only the target E.coli contain the trans-activating RNA (key) to activate the expression of the virus developmental genes. Once the genes are activated, the target cells will go through the lysis process. Meanwhile, non-target cells won’t be affected by the virus because the non-target cells lack the key to activate the expression of the virus developmental genes.

Caltech Riboregulator references

  1. Isaacs, Dwyer DJ, Ding C, Pervouchine DD, Cantor CR, Collins JJ. "Engineered riboregulators enable post-transcriptional control of gene expression"

TU Delft 2009

Abstract: TU Delft 2009 wanted to build an improved cell to cell communication system. They wanted to construct an E.coli strain that can pass GFP signals through conjugation only once. When “initiator cells” which contains the “signal plasmid” for the communication system, is added to a cell culture, the initiator cells (donor cells) conjugate with other cells “non-initiator” cells in the culture. During the conjugation, the signal plasmid is transferred to the recipient cells. The signal plasmid encodes for an entry exclusion protein, an endonuclease, and GFP. It has a restriction site of the endonuclease. The entry exclusion protein prevents backward conjugation, the endonuclease and the restriction site can reset the cell back to off state, and finally GFP can a reporter to visualize where the signal is. The expression of endonuclease should be delayed because cells, which just received the signal plasmid, require time to conjugate and pass down the signal plasmid to the recipient cells. TU Delft 2009 tried to solve this problem by introducing a delay device. The riboregulator system was used for their RNA-based delay system. By having another level of regulation between the reception of signal and expression of self-destructing protein (e.g. endonuclease), Delft 2009 believed that there could be delayed expression of the self-destructing protein. TU Delft 2009 also designed an algorithm called Ribo Key & Lock Generator that can generate sequences for lock and key riboregulators when RBS sequence is used as an input.

TU Delft Riboregulator references

  1. Smolke C, 2009, It's the DNA That Counts, Science, 324:1156-1157.
  2. Sprinzak D and Elowitz M, 2005, Reconstruction of genetic circuits, Nature, 438-24:443-448.
  3. Isaacs F, Dwyer d and Collins J, 2006, RNA synthetic biology, Nature Biotech., 24:545-554.
  4. Anderson C, Voigt C and Arkin A, 2007, Environmental signal integration by a modular AND gate, Molecular systems biology, 3-133:1-8.
  5. Isaacs F, Dwyer d and Collins J, 2004, Engineered riboregulators enable post-transcriptional control of gene expression, Nature Biotechnology, 22-7:841-847.

Groningen 2011

Abstract: “Our project aims to design a genetic device able to count and memorize the occurrences of an input signal. We achieved this by utilization of auto-inducing loops, that act as memory units, and an engineered riboregulator, acting as an AND gate. The design of the device is modular, allowing free change of both input and output signals. Each increase of the counter results in a different output signal. The design allows implementation of any number of memory units, as the AND gate design enables to extend the system in a hassle-free way. In order to tweak bistable autoinducing loops we need a very fast and robust method for characterizing parts. For this we have created a genetic algorithm that will enable us to find parameters of the parts used in the design. It also allows the combination of data from multiple experiments across models with overlapping components.”

Groningen Riboregulator reference

  1. Isaacs, Dwyer DJ, Ding C, Pervouchine DD, Cantor CR, Collins JJ. "Engineered riboregulators enable post-transcriptional control of gene expression"

K.U. Leuven 2009

Abstract: 'Essencia coli' is a vanillin producing bacterium equipped with a control system that keeps the concentration of vanillin at a constant level. The showpiece of the project is the feedback mechanism. Vanillin synthesis is initiated by irradiation with blue light. The preferred concentration can be modulated using the intensity of that light. At the same time the bacterium measures the amount of vanillin outside the cell and controls its production to maintain the set point. The designed system is universal in nature and has therefore potential benefits in different areas. The concept can easily be applied to other flavours and odours. In fact, any application that requires a constant concentration of a molecular substance is possible.

Melbourne 2008

Abstract: This year Melbourne iGEM competition team seeks to build a temporal controller in E. coli. The idea is to build a system, which is modular, has all components in the form of biobricks, and expresses gene(s) at a specific time in a sequential manner. In this study, we show the design, modeling and some experimental results towards a proof of principle of the system. The design uses the leverage of existing biobricks of red light bacterial photography system, positive feedback loops and riboswitches. We propose that the architecture presented should scale well with increasing number of genes to be temporally regulated. It is anticipated that such system will be useful in metabolic engineering because enzymes can be turn on and off in a sequential manner.

Melbourne Riboregulator reference

  1. Bauer et al. (2006). Engineered riboswitches as novel tools in molecular biology. J Biotechnol vol. 124 (1), 4-11.2.
  2. Ray, S.K. (2004). Riboswitch: A new mechanism of gene regulation in bacteria, Current Science, Vol. 87 (No. 9).
  3. Isaacs et al. (2004). Engineered riboregulators enable post-transcriptional control of gene expression, Nature Biotechnology, 22 (7), 841-847.

Peking 2007

Abstract: Our projects concern with the ability for bacterial cells to differentiate out of homogeneous conditions into populations with the division of labor. We aim at devices conferring host cells with the ability to form cooperating groups spontaneously and to take consecutive steps sequentially even when the genetic background and environmental inputs are identical. To break the mirror in such homogeneous condition, we need two devices respectively responsible for temporal and spatial differentiation. The implementation and application of such devices will lead to bioengineering where complex programs consisted of sequential steps (structure oriented programs) and cooperating agencies (forked instances of a single class, object and event oriented) can be embedded in a single genome. Although this "differentiation" process resemble the development of multicellular organism, we tend to use a more bioengineering style analogy: assembly line. Or maybe after some years from now, this will not be just an analogy.

Victoria BC 2009

Abstract: This project explores some of the ways that the secondary structure of messenger RNA can be used to control the rate of protein expression. The 32oC ribothermometer made by the 2008 TUDelft team will be coupled to fluorescent proteins to visually confirm temperature-dependent translation. The "ribolock" made by the 2006 Berkeley team will be tested at various temperatures to determine if it could double for use as a ribothermometer. Finally, a proof-of-concept NAND logic gate will be constructed: a ribolock will be used to interpret two concurrent environmental signals into an on/off control for mCherry output.


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