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

Introduction to riboregulators

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.

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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

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

Usage of riboregulator by different iGEM teams

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 Berekeley 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).

UC Berkeley 2006

Berkeley riboregulator page

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

Calgary riboregulator page

Abstract: The project we selected was to design and build a biomechanical printer; composed of a two dimensional plotter equipped with a red laser, software to translate computer images into instructions for the plotter, and E. coli cells engineered to respond to the laser light. Bacteria are spread in a solid lawn on the plate, or mixed in the media before pouring the plate. The response triggered by this biological circuit will produce beta agarase, an enzyme which degrades the agar polymer that the cells rest on.

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.

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.

Caltech 2007

Caltech riboregulator page

Abstract: 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?

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.

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.

Caltech Riboregulator references

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

Delft 2009

Delft Riboregulator page

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

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

Groningen Riboregulator page

Abstract: 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.

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 2008

K.U Leuven 2008 Riboregulator page

Abstract: 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.

K.U. Leuven 2009

K.U Leuven 2009 Riboregulator page

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

Melbourne 2008 Riboregulator page

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

Peking 2007 Riboregulator page

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.