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.

In order to connect different BioBricks, our network requires four major types of components:

• Input elements
• Transmitter molecules
• Logic gates
• Output elements

These elements can be combined to build up a molecular network (see illustration). Each input molecule (such as a BioBrick) produces a unique transmitter 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). These logic gates produce other transmitter molecules, which can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected by transmitters. The last "layer" of logic gates finally allows to active/inactive output elements, such as BioBricks.

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 determine what type of molecules can perform the required functions. We decided to use RNA both as transmitter molecules and for constructing logic gates. Several advantages result from the utilization of RNA as the central element:

• During the last years, many Biobricks were designed that are sensitive to various chemicals and substances. These BioBricks often function as a transcription factor that binds to a specific DNA sequence and consequently would be capable to produce a specific transmitter RNA molecule. Thus, in principle each BioBricks which involves transcription can be used integrated in our circuits/networks
• Since all logic gates would be capable to produce RNA (since the transmitter is RNA), they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as output of our circuits/networks.
• If RNA forms both, the transmitter molecule and the logic gates, they can specifically interact by RNA-RNA interaction, which is highly predictable compared to protein interactions. This allows to generate a library of transmitters and gates in silico.
• RNA production is fast and energy saving for a cell - since the complete faulty wiring of our circuits/networks (the interactions of logical gates) would be on the level of RNA-interaction and thus on a transcriptional level, translation and resource consuming protein production would one be involved at the last layer.
• As the half-time of RNA can be rather short, transmitter RNA will not accumulate within the cell and it is therefore less likely for the system to become saturated.

Generally speaking, our logic gates are to posses the following characteristics:

• Logic gates, such as AND, OR and NOT, have to be implemented by RNA-interaction based principles (see How to connect BioBricks).
• All logic gates have to recognize corresponding transmitter RNA´s and, in response, produce an output transmitter molecule.
• Logic gates should follow a basic design rule, in such a way, that their creation can be automated in silico.
• The response efficiency of logic gates toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness, sensitivity and comparable molecular concentrations to be functional
• the whole system should be able to function in vivo - therefore, all parameters where optimized to 37 °C and a e.coli environment

In order to build a logic gates for our bioLOGICS system we will first create a simple toggle switch. A toggle switch can be activated by one transmitter RNA and produce an output transmitter RNA. In contrast to a logic gate, a toggle switch does not perform logic operations. However by combining toggle switches, logic gates can be created. The following text will first describe how the developed toggle switch works and secondly, how logic gates such as AND/OR/NOT can be created by based on these toggle switches.

Toggle Switch

• target site
  

The target 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 RNA-production (transcription), a "switchable" transcriptional terminator is suitable for this purpose. The principle idea of our target site relies on such systems occurring in nature. We used in silico synthetic RNA-engineering to modify the Antitermination principle of two natural system: Attenuation in e.coli and tiny abortive RNA´s of T7-phage. Future plans will also work with Synthetic Terminators, which will retrieve additional informations what drives the process of Termination. To highlight and illustrate the functional principle of our switches, only the part of the terminator which is involved in interacting with a transmitter molecule and which is responsible for shifting between "on" and "off" state is called target site. The remaining terminator sequence is called often called terminator in the following, even if both, target site AND terminator equal the terminator structure occurring in nature.
The sticking point of our switches is the fact that they all consist of THE SAME target site. Having found one functional "switchable" terminator will therefore allow almost unlimited upscaling. This is the main difference to previous works on this field, which always required developing a new shifting principle for each switch.^[12][13][14] Beside the extendability, this principle provides a comparable on/off shifting rate, which avoids complex concentration dependent fine tuning of interacting molecular circuits.
Another special thing is the specific accessing of our toggle switches is separated from the target site, by providing an additional recognition site.

• recognition site
  

It defines the specific accessibility of a respective toggle switch by an transmitter molecule. Therefore, a unique recognition element is assigned to each switch. This allows to arrange and interconnect numerous of these switches in a specific logical order, without changing the target site, comparable to wires connecting many transistors.
The recognition site is implemented by putting a random sequence with arbitrary length (has to be optimized, compare in silico design and Modeling ) upstream of the target site.

Transmitter RNA´s, input and output of bioLOGICS toggle switches

Transmitters present the "trigger" to shift switches between the "on" and "off" state. They requires the ability to change the terminators secondary structure and cause antitermination, BUT only if a special recognition site is detected. Thus, each signal consists of a trigger site, interacting with the switches trigger site and an identity site, interacting with the toggles recognition site. Practically, the shift between antitermination and termination is induced by a complementary RNA-sequence, influencing the terminators secondary structure. But in contrast to previous approaches on this field ^[12], we introduced the described synthetic trigger site in such a manner it is not able to change the terminator´s state on its own, but only in combination with the identity site, which is complementary to the recognition site.

The challenge is to arrange and optimize these elementary building blocks thermodynamically, that a trigger site is only able to switch in combination with its respective identity site. This was done by in silico design using NUPACK, presented in section in silico design.

Putting it all together: the switching process

Rectangles present the composition of our functional units on the level of DNA. Fringed lines represent RNA produced by RNA polymerase. The stem loop structure depicts the switchable terminator. Terminator and target site are illustrated in blue and turquoise, respectively. Recognition sites are indicated in different colors, in this case red for the input transmitter and green for the output transmitter.Each toggle switch and or later logical unit has to be flanked by a promotor and another constitutive terminator, to allow RNA-production by RNA-polymerase in a proper way.

On the other hand, in the presence of a input transmitter, this small functional RNA inhibits the stem loop formation by complementary base-pairing and hence avoids termination of transcription. In detail, the identity site (red part on transmitter) binds the recognition site (red part on switch) and serves as toehold, which will thermodynamically allow the trigger site (turquoise part on transmitter) to perform a strand displacement and open the stem loop structure, allowing the polymerase to read through and form the output RNA.
Summing up, we use can use this concept to create a switch that can be toggled by a transmitter RNA molecule and in response, is able to produce another transmitter RNA.

Main challenges are

• to find a suitable terminator construct and design a complementary trigger unit, which is only functional in combination with a specificity site - meaning an optimization of the thermodynamically parameters (see in silico design)
• to investigate whether the signal/switch interaction reaction is really on a timescale

to be competitive to terminator formation - meaning an comparison of kinetic parameters (see Modeling page)

• to proof antitermination can be also be caused by synthetically RNA-interaction (see Antitermination in nature and in vivo and in vitro measurements )

The bioLOGIC toggle switch with regard of logical operations

As described, each switch can be accessed by a specific RNA-transmitter molecule, illustrating the input. In turn, another RNA-transmitter molecule will be produced if the switch shifts its state. This output transmitter of one switch can serve as input transmitter for the next toggle switch by meaningful selection and design of the respective recognition sites. This easily allows arranging several switches in specific sequences and faulty wiring - the corner stone of a logical network.

To ease the building of logical networks, applying mathematical logics, e.g. Boolean logics like in computational science would be worthwhile. It is possible to establish general Boolean operators with our switches and thus build "logical modules". Since AND/OR/NOT are the most simple logic operations which can be implemented with the presented toggle switches, and all remaining operations can be expressed by these three operators[ZITAAAT Wiki oder so)we exemplary designed them.

*AND consists of a parallel circuit of two switches

*OR is implemented by connecting two switches in series

*NOT is more complex to explain. In principle, it consists only of one toggle switch which contains its respective signal molecule intrinsic, so via intramolecular interaction, antitermination is the initial state. The signal is composed of the same components as usual to allow interconnection with other logic gates.

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

attenuation principle

A random sequence was derived from xxx. A complementary sequence, reaching within the terminator´s stem loop was stepwise shortened to find the length, where the formation of the terminator is thermodynamically favored compared to the strand displacement by the signal. The trigger sequence was defined by selecting the shortest unit which still is able to "destroy" the stem loop. Subsequently, the trigger unit was tested in regard of not being able to resolve the stem loop on its on. As table xxx illustrates, the terminator is thermodynamically favorite toward the trigger unit, but in combination with the specificity site, binding becomes possible.

Pairing probabilities of the His-Terminator based toggle switch simulated by NUPACK secondary structure analysis tool	Illustration of the His-Terminator based toggle switch secondary structure, generated by NUPACK utility tools
Pairing probabilities of the His-Terminator based toggle switch in presence of the respective transmitter RNA, simulated by NUPACK secondary structure analysis tool	Illustration of the His-Terminator based toggle switch secondary structure in presence of the respective transmitter RNA, generated by NUPACK utility tools
Pairing probabilities of the His-Terminator based toggle switch in presence of the transmitter RNA´s trigger site only, simulated by NUPACK secondary structure analysis tool	Illustration of the His-Terminator based toggle switch secondary structure in presence of the transmitter RNA´s trigger site only, generated by NUPACK utility tools
Pairing probabilities of the His-Terminator based NOT-gate in presence of the transmitter RNA´s identity site only, simulated by NUPACK secondary structure analysis tool	Illustration of the His-Terminator based NOT-gate secondary structure in presence of the transmitter RNA´s identity site only, generated by NUPACK utility tools

NOT Gate

Pairing probabilities of the His-Terminator based NOT-gate simulated by NUPACK secondary structure analysis tool	Illustration of the His-Terminator based NOT-gate secondary structure, generated by NUPACK utility tools
Pairing probabilities of the His-Terminator based NOT-gate in presence of the respective transmitter RNA, simulated by NUPACK secondary structure analysis tool	Illustration of the His-Terminator based NOT-gate secondary structure in presence of the respective transmitter RNA, generated by NUPACK utility tools
Pairing probabilities of the His-Terminator based NOT-gate in presence of the transmitter RNA´s trigger site only, simulated by NUPACK secondary structure analysis tool	Illustration of the His-Terminator based NOT-gate secondary structure in presence of the transmitter RNA´s trigger site only, generated by NUPACK utility tools
Pairing probabilities of the His-Terminator based NOT-gate in presence of the transmitter RNA´s identity site only, simulated by NUPACK secondary structure analysis tool	Illustration of the His-Terminator based NOT-gate secondary structure in presence of the transmitter RNA´s identity site only, generated by NUPACK utility tools

tiny abortive principle

ubiquitous terminators

Considering the high complexity of in vivo measurements compared to other experimental challenges, a robust and easy to handle test system for PoPS-based devices is desirable. As described in Experimental design, we used fluorescent proteins: eGFP to normalize the input and a RFP or mCherry to measure the output. Our first t attempt, using the screening plasmid pSB1A10 yielded no interpretable results. Switching the fluorescent protein to mCherry did not work either, but after several experimental setups we determined a transcriptional problem causing no reporter protein expression regardless of the inserted part. Thereby we demonstrated the screening plasmid pSB1A10 to be malfunctioning. Finally a new design based on pSB1A10 lead to a functional and robust screening system. A second promoter with identical induction properties inside the BioBrick cloning site enforces transcription of the PoPS-based device and the mCherry output.

Exemplary, the graph below on the right shows the positive control, induced and uninduced at OD₆₀₀=0.7 followed by 16 h incubation at 25 °C. Clearly visible are eGFP and mCherry fluorescence in the induced samples. The uninduced control showed no fluorescence at all, demonstrating the PBad promoter to be tight and providing no basal transcription. A major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and toggle switches in particular easy and robust.

Bacteria containing positive control

Emission spectra of induced (green/red) and uninduced(black) positive control BBa_K494002 ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm

Due to the time limitations of the iGEM completion we had to focus our efforts on few toggle switches after designing the screening system. Since nobody does it better than nature, we choose the HisTerm as well as the TrpTerm. Both switches are based on known natural [[2]]. Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficancy.^[12] The exemplary measurement below on the right confirms the high terminator efficiency. In fact, we could not detect any mCherry fluorescence in any cells containing the HisTerm. Even induction of the corresponding signal transmitter RNA via IPTG did not alter the Terminator efficiency. Again time was the limiting factor and prevented us from testing more than one corresponding signal. Thus, the results are insufficient to disprove the functionality of the HisTerm or our concept in general.

Bacteria containing HisTerm

Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing HisTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm

Attaining only 90% terminator effiecency, the natural Trp [[3]] is known be less effective than the HisTerm.^[13] The graph on the right depicts the to our designed TrpTerm characteristic efficiency of about 40 %, notably below the natural standard. Allowing 60% transcription in the “off” state excludes the TrpTerm from possible candidates for a scalable network of logic gates. Thus the TrpTerm is inoperative as intended, but may still be useful in other contexts. Similar to the HisTerm, the TrpTerm also did not react to the induction of the corresponding signal.

Bacteria containing TrpTerm

Emission spectra of induced and uninduced screening plasmid BBa_K494002 containing TrpTerm ; green: eGFP fluorescence ex: 501 nm, red: mCherry fluorescence ex: 587 nm

Making use of our improved screening system we also carried out some in vivo kinetic measurements in addition to the end-point measurements above. In contrast to the in vitro experiments we did not obtain significant results for the chracterization of our toggle switches. As the switching process is many times faster than protein synthesis our in vivo kinetics include the synthesis of mCherry as well as its maturation. Therefore we centered our attention on end-point experiments. For more information browse the lab book.
Considering our in vivo measurements, neither of the tested switches showed any effect regarding the signal induction. But due to the small number of tested switches and signals this can hardly be regarded as disprove of concept. In particular in light of the recent findings by Sooncheol proving antitermination in principle using a T7 system.^[14]

in vitro translation

in vitro transcription

malachite green assay

RNA-PAGE