Team:TU Munich/Project

From 2010.igem.org

Revision as of 20:57, 23 October 2010 by Hartlmueller (Talk | contribs)

Navigation:

Home →  Project

iGEM MainPage

Contents

bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation


Vision

Until today, 13.628 biobrick sequences[1] have been submitted to partsregistry, thereof 102 reporter units, 12 signaling bricks and xx sensing parts. Since there, people are trying to arrange these single biological building blocks in such a manner that allows producing special biotechnological products (metabolic engineering), developing biological sensory circuits (biosensors) and even giving microorganisms the ability to react on multiple environmental factors and serve both as disease indicator and drug. These examples and further promising ideas were implemented on previous iGEM-competitions.[2][3][4]

The idea of combining the outcome of several iGEM competitions to construct complex synthetic biological systems falls at the last hurdle - the fact, that each team uses a different principle how to access and functionally connect the respectively used biobricks. For example, it is a major challenge to create a system that uses several sensoring BioBricks from different iGEM-teams which in turn regulates reportering BioBricks from various teams. In order to combine and fully take advantage of these promising projects, our vision is to develop an adapter that allows interconnecting any arbitrary biobricks on a functional level. Such a system easily allows to setup sensor-reporter circuits and interconnect them to complete biological chips... A further step towards artificial cells.

Generally speaking, the above descirbed adapter has to meet the following requirements:

  • Universality
The adapter has to be compatible to as many BioBricks as possible. This objective will guarantee that a large number of BioBricks can be connected.
  • Scalability
Once the basic design of the system is established, the construction of the system is supposed to be automated in silico. This way it will be possible to create an adapter connecting a large amount of BioBricks.
  • Biological orthogonality
Interfering with cellular components has to be as low as possible in order to avoid unwanted and perturbing side effects.
  • Logic
The adapter is supposed to not only associate different BioBricks, but to functionally connect BioBricks in a precisely determined manner (including operations such as AND/OR/NOT).


Several biological logic units, devices and circuits have been developed so far[5], but to our knowledge, none was shown to meet all requirement listed above.

Implementation

To functionally connect BioBricks there are several possibilities including genetic switches, riboswitches and direct protein-protein interactions. We investigated several hypothetically principles, and decided to focus our practical work on the development of a RNA-RNA interaction-based switch. These switches are capable of changing between two states, a state of antitermination and termination, and make use of highly-specific RNA-RNA interaction. In principle such a switch can fulfill all requirements mentioned previously. The following text clarifies how these switches work in detail.

Read more

How to connect BioBricks

Our adapter is a system, that activates or disables BioBricks (output BioBricks) in response to the presence of other Biobricks (input Biobricks). Our appraoch uses a molecular network to put this into practise and consists of four major elements:

Examples of a molecular network. Molecules are indictated as lines, whereas black represents input molecules, red indicates transmitter molecules, switches are green and labeled with their type and output molecules are drawn blue.
  • Input elements
  • Transmitter molecules
  • Logic gates
  • Output elements

These elements can be combiined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a transmitter molecule that is unique for this input molecule. All transmitters belong to the same type of molecule and share a common design. However each transmitter molecule can only interact and activate a certain subset of logic gates. In other words, logic gates have to recognize and bind the corresponding transmitter molecules. Depending on the type of the logic gate (AND, OR or NOT[6]), an output molecule is only created if both transmitter molecules are present (AND), at least one of two transmitter molecules is present (OR) or if no transmitter is present at all (NOT). Consequently a certain subset of output molecules are generated accoring to the presence of transmitter molecules, thus also accorind to the corresponding BioBricks.

Computer vs. molecular netowrk
Logic gates in a molecular network can be compared to transistors used in a computer where billions of transistors are incorporated. on a computer chip, these transistors are connected by wires in such a way that the output of one transistor only acts on a subset of other transistors. However, spatial connection are not possible in a living cell. The wiring within a cell relies on specific interaction between the transmitter molecule and the correcponding logic gates.

Again, these output molecules are transmitter molecules and can in turn interact with another subset ("layer") of logic gates. In theory many layers of logic gates can be connected by transmitters. The last layer of logic gates has to generate transmitter molecules, that do not interact with logic gates but induce output molecules, such as Biobricks, instead.

Computer vs. molecular netowrk
Logic gates in a molecular network can be compared to transistors used in a computer where billions of transistors are incorporated. on a computer chip, these transistors are connected by wires in such a way that the output of one transistor only acts on a subset of other transistors. However, spatial connection are not possible in a living cell. The wiring within a cell relies on specific interaction between the transmitter molecule and the correcponding logic gates.

Summarizing, the network established a connection between input BioBrick and output BioBrick in a functional manner.
Having addressed the basic layout of the molecular network, the next step is to decide what type of molecules can perform the required functions. For our project we used RNA for transmitter molecules as well as for logic gates. Several advantages result form the utilizationof RNA as the central element:

  • During the last years, many Biobricks were designed that are sensivtive to various chemicals and substances. These BioBricks often function as a transcription factor that bindes to a specific DNA sequence and consequently are capable to produce a specific transmitter RNA molecule.
  • If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact with each other. Furthermore, the



  • Logic gates
A logic gates is a small unit, that generates an output only under certain conditions. Depending on their type (AND, OR or NOT) an output is only created if both input are present (AND), at least one of two inputs is present (OR) or if no input is present at all (NOT.
To build up a network containing several logical gates, it is important that the output generated can serve as an input for another gate. That is, the output and input not only have to be the same of molecule but they also have to share the same functional design.

How is it really working - The smallest locial unit - establishing an equipollent to an electronic

transistor unit

THe main goal is to provide universality, so input and output on same basis, RNA, transcribiton, sin

THe main goal is to provide


switch

  • switching element

The switching unit is the functional core element of our switches, allowing a shift between an "on" and "off" state. Since we work on the level of transcription, a "switchable" transcriptional terminator is suitalbe for this purpose.

Read more

The principle idea of our switching unit relies on such systems occuring in nature. We focus on two system: - Attenuation-Operon - tiny abortive RNA´s

Close

The special thing about it is all of our switches consist of THE SAME switching unit, so having found one functional "switchable" terminator will allow almost unlimited upscaling. This is the main difference to previous works on this field, which always required to develop a new shifting principle for each switch. Beside the extendablity furhter advantages are we can provide a comparable on/off shifting rate. This makes our switches more similar to transistors than ...

Practically, the shift between the two states is induced by complementrary RNA-sequences, influencing the terminators secoundary structure. To seperate

this switching element from the specific accessiblity, we designed a synthetic switching unit relying on NUPACK simulations, which is not able to change the terminators state on its own, but only in combination with a specific recognition site:

  • recognition site

It defines the specific access of one of our switches by an input molecule. Therefore, a unique

recognition element is assigned to each switch. This allows to to arrange and interconnect numerous

of these switches in a specif logical order, comparable to wires connecting different transistors.

Read more

It is implemented by simply putting a random sequence with arbitrary lenght (has to be optimised) in front of the switching unit. Christoph: thermodynamisch kinetisch usw reicht dass um zu schalten?? xxx possbilites to build unique switches. (blacklist, etc)

Close

signals, the inputs and outputs of the bioLOGICS switch

Signals present the "trigger" to shift switches between on and off. It requires the ability to change the terminators secondary structure BUT, only if a special recognition site is detected.

Read more

Thus, each signal consists of a trigger unit, interacting with the swichtes´ switching unit and a specifity site, interacting with the terminators recognition site.

The challenge is to arrange and optimise these elemantary building blocks, that a trigger unit is only able to switch in combination with its respective specifity site.

Close

The bioLOGIC switch with regard of logical operations

As described, each switch can be accessed by a specific RNA-signal molecule, illustrating the input. In turn, another RNA-signal molecule will be produced if the switch shifts its state, now beeing the output of one switch and at the same time, a possible input for the following switch or several ones. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.

To easen the building of logical networks, applying mathematical logcis, e.g. Boolean logics like in computaltion science would be worthwile. It is possible to establish general Boolean operators with our switches and thus build "logical modules".


Read more

Since AND/OR/NOT are the most easy logic operations modularisable with our switches and can substitute all remaining operations, we exemplarly designed them.

    • AND consists of a parallel circuit of two switches

AND.png
    • OR is implemented by connecting two switches in series
      OR.png
    • NOT contains its respective signal molecule intrinsic, so via intramolecular interaction, the antitermination is the initial state. the signal composed of the same components as usual but its sequence is complementary.
      NOT.png

Close

Evaluation

To evaluate functionality of our molecular switches, we developed several in vivo and in vitro assays and relied on existing assays. Thus we don't want to develop additional biobricks, we rather want to establish a platform to realize the full opportunity of the biobrick system. This would enable people easily setting up sensor - reporter circuits AND interconnect them to complete biological chips... the way to real artificial cells and synthetic biology. (zu krass?)

Network construction

Designing complex biological networks based on either traditional protein engineering or our new bioLOGICS is still a complex task. We developed a software which allows the fast construction of a bioLOGICS based networks.
To read more about this, look at our Software page

Close

Concept

The basic principle of our switches are short RNA sequences, and the scientific idea shares similiarities with the principle of antitermination, but also inherits a completely new way of RNA based transcription regulation. We used three-dimensional structure predictions and thermodynamic calculation to develop a set of switches - about 50 nucleotides - and signals - about 20 nucleotides. The switches form a stem loop causing transcription termination, which can be resolved upon binding of the specific signal. On/off switching can therefore be easily controlled by signal avaiability and provides a new concept to control gene transcription.

Read more

Our switches are based on the principle of transcriptional antitermination. Transcription can be cancelled by the formation of a RNA stem loop in the nascent RNA-chain, which then causes the RNA polymerase to stop transcription and fall off the DNA. We use sequences as transcriptional switches, that are calculated to be capable of stem loop formation.

Novel switches based on RNA-RNA interaction

We plan to control the termination at these switches with a small RNA molecule (our RNA "signal") that is complementary to a part of the stem loop forming sequence. This small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids transcription termination. The signal is composed of two parts: While the first part provides specifity (recognition site), the second part causing stem loop disintegration (functional core) can in principle be the same for all bioLOGICS. Therefore variation of the first part allows the construction of an endless number of switches. The functional core causing stem loop disintegration is based on a working system (see attenuation) established by nature. Different stem loops were tested in this effort: Regulatory parts from the E. coli trp-operon, his-operon and one based on previous iGEM-work.
The initial signal can be provided by various metabolic compounds and stimuli and the output signal can be anything DNA-coded, too. In the last years, many working sensory systems were submitted to the Partsregistry. Those parts can now be utilized as inputs for our network. BioLOGICS provides a new way to use the whole potential of iGEM distributions connecting different parts for totally new applications.
The major advantage over conventional protein based devices and even riboswitches is the small size of our basic units, its easy construction, huge variability and easy upscaling to complex networks in cells. Furthermore introduction of an RNA network into E. coli is especially easy - all informations can be coded on one plasmid. In comparison to protein networks, the elegance of RNA based networks lies in its functionality, simplicity and predictability. Since RNA-RNA interactions are highly predictable and since our system is based on the variation of one principle in many switches, simulations of our networks are much more accurate than those of comparable systems currently available. Incorporation of established Biobricks allows a high diversity of input and output signals providing a whole new level of cell control.

Close


Results

Every network starts with a basic unit. While our declared aim is to enable networks allowing fine-tuning of gene expression beyond the regular on/off, exploring such an on/off switch/signal pair is the first step towards a functional network. We constructed several units and tested their efficiency, robustness and reproducibility in vivo, in vitro and in silico. Furthermore we developed a software which allows easy constructions of networks delivering a ready network. Conclusive elaboration of a few first RNA-based logic units is the major contribution of our iGEM team.

References

[1] http://partsregistry.org/cgi/partsdb/Statistics.cgi [2] https://2009.igem.org/Team:Imperial_College_London/M1 encapsulation [3] https://2009.igem.org/Team:TUDelft [4] https://2008.igem.org/Team:Heidelberg [5] Smolke and so on.... [6] http://en.wikipedia.org/wiki/Logic_gate#Symbols