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

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

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

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: 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

Caltech riboregulator page

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

TU Delft Riboregulator page

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

Groningen 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.”

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

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: Melbourne 2008 team seeked to build a biological clock that can count up when it is "ticked" by input light pulse signals. As the signal "ticks" the clock, the clock changes color and observer can use the color or combination of colors to tell what "time" it is. Their team built a linear clock which can be used to keep track on what metabolites are present and what are being produced in the biological system.

In their construct, ribolock 1(CR) prevents translation of gene Y and in the presence of ribokey1(TA), gene Y is translated to activate transcription of another ribokey2(TA) which can open up the loop of another ribolock2(CR). Although they claimed to design and construct some of the parts including key3C and lock3d, no characterisation result was found regarding these CR and TA.

Melbourne Riboregulator references:

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: Peking 2007 project concerned with the ability for bacterial cells to differentiate out in homogeneous conditions. They aimed to build 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 . In order to achieve this, they needed two devices responsible for temporal and spatial differentiation.

To meet the need of spatial differentiation in an assembly line, the system of hop-count conjugation for cell-cell communication was developed in E. coli. The signals transferred are represented by DNA sequence, and record the times of transfer by losing tandem region at every conjugation event. To construct this system, tandem oriT pairs are inserted into signaling plasmid, and relaxase responsible for initiating conjugation events are removed from natural conjugation system and (attaching a terminator) inserted between these oriT pairs. At each single conjugation event, with the expression relaxase, the corresponding tandem oriT pair is lost with the rolling-circle replication. Sequential conjugation and deletion of the different oriT pairs can be achieved by fine control of relaxase expression. To report the ending of tandem conjugation process, a reporter gene is attached after the last oriT pair, and hence expresses after all conjugation events happened.The resulting tandem signaling conjugation process will allow them to develop networks of bacteria capable of sequentially assembling tasks.

The riboregulator system was incorporated in Into their circuit to provide an interface for interaction between their conjugation system and general genetic devices to realize the goal of assembly line. According to the data that they measured, they concluded that the open ratios of Key1 -Lock1 and Key 2-Lock1, the open ratio of key1-Lock1 was 3.3 times higher than Key2-Lock1.

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.