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 as well as bind the corresponding transmitter molecules and are capable of producing a new output transmitter molecule. Depending on the type of the logic gate (AND, OR or NOT^[6]), an output transmitter is only created if both input transmitter molecules are present (AND), at least one of two input transmitters is present (OR) or if no input transmitter is present at all (NOT). Once a logic gates has produced a new output transmitter, these transmitters can in turn address another subset ("layer") of logic gates. In theory many layers of logic gates can be connected this way allowing the creation of large networks. Until this step, various transmitter molecules might have been produced. But in order to create a Biobrick output, the last layer logic gates finally generates transmitter molecules that will not active logic gates, but will rather interact with the cell metabolism to produce a BioBrick responds. In other words, the last layer of transmitter molecules is capable of regulating BioBrick formation.

Summarizing, the network established a connection between input BioBricks and output BioBricks 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 BioBrick which involves transcription can be integrated in our network.
• Since all logic gates are capable of producing transmitter RNA, they can also produce functional mRNA encoding any protein. This means, each BioBrick consisting of protein or RNA can be produced as an output of our network.
• 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. Such a library is essential for the creation of large networks.
• RNA production is fast and energy saving for a cell. Consequently, operating a network that only produces RNA rather than proteins will also be faster and more efficient for the host cell. Since our logic gates are based on transcription, translation and resource consuming protein production will only be required at the very last step.
• 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 their 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 a logic gate toward a transmitter molecule should be comparable for all logic gates to provide calculable robustness and sensitivity. This will ensure comparable molecular concentrations and functionality of large networks.
• The system has to be designed for in vivo utilization at the first place. As a reference we always assumed a temperature of 37 °C and an E. coli environment.

Switch

Roughly speaking, a switch can be regarded as an enhance transcriptional terminator. The enhancement can be described easier by dividing a switch into its components:

• Target site
  

The target site 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. By allowing or preventing formation of a transcriptional terminator, that is by switching between termination and antitermination it is possible to represent an "off" and an "on" state, respectively. Therefore, the target site is the 5' ending of the terminator and is required for a stable terminator formation. It should be noted that this principle was also observed in nature.

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 terminator in the following, even if both, target site and terminator build up the terminator structure occurring in nature.

The important aspect of our switches is the fact that all switches will hold the same identical target site. Therefore having found one functional "switchable" terminator, will allow almost unlimited upscaling since this terminator can be used for a large library of switches. This is the main difference to previous works done on this field, which always required developing a new shifting principle for each switch.^[12][13][14] Beside this scalability, this principle provides a comparable on/off shifting rate (responds function) for all switches, avoiding complex fine tuning of molecular networks.

To sum it up, the target site, allows to switch between an "on" and "off" state. But so far, the switch is not capable of performing specific interaction with transmitter molecules. This is where the recognition site comes into play.

• Recognition site

The recognition site defines which transmitter molecule can actually interact with the switch. Therefore, a unique recognition site is generated for each switch and is positioned right upstream of the target site. In principle the recognition can be any random sequence as long as it remains unique within the molecular network.

Summing up, the recognition site allows a specific interaction between switches and transmitter molecules. Once this interaction is formed, an interaction between the transmitter and the target will actually switch the state of the terminator. This allows the specific arrange and interconnection of numerous of these switches by transmitter molecules, without changing the target site. Comparable to wires connecting many identical transistors, our target site remains the same.

Transmitter RNA´s

As desccribed above, transmitter RNA's are the input and output of bioLOGICS switches (compare How to connect BioBricks). These transmitters are short ssRNA molecules representing the "trigger" to shift switches between the "on" and "off" state. To fulfill this role, they need to posses the following properties:

• A transmitter may only interact with certain switches. That is, a transmitter has to find the corresponding recognition site of a switch.
• Once an interaction is established between a transmitter and a switch, a transmitter as to be capable of changing the secondary structure of a terminator and thus cause antitermination.

Again, these two properties are fulfilled by two comnponents of the transmitter:

• Identitiy site

This site is capable of forcing an interaction between the transmitter and the switch. Therefore it is complementary to the recognition site of this switch. As the recognition site is unique within a network, so it the identity site. However, the single identity site is not capable of changing the state off the switch. That is were the trigger site comes into play.

• Trigger site

Once an interaction is created by the identity site, the trigger site is capable of actually shifting the switch since it is complementary to the target site of the switch. To fulfill this role, it is placed upstream at the 5' end of the identity site. As the target site is the same for all switches, the trigger site is the same for all signals. Therefore it is important, that similar to the identity site, a trigger site cannot function on its own. That is, a single trigger site cannot shift the state of a switch without the help of an identity site.

Summing up, we applied the principle introduce for the switches to the transmitter molecules. In contrast to previous approaches on this field ^[12], we introduced the described synthetic trigger site in such a manner that it is not able to change the state of the terminator on its own, but only in combination with the identity site. So 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

The functional principle of the designed switches is illustrated in the figure. The switch is positioned on DNA upstream of a desired output transmitter. So in the absence of a triggering transmitter molecule, transcription will be canceled by the formation of a RNA stem loop in the nascent RNA-chain. This will cause the RNA polymerase to stop transcription and fall off the DNA and consequently no output RNA will be produced. This process only relies on rho-independent termination. 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 up the stem loop structure. Consequently the polymerase can read all the way through and form the output RNA.
Summing up, we 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.

From switches towards bioLOGICS logic gates

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 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 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 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.

Once the objective mentioned above is accomplished, these basic RNA/RNA-interactions have to be modified in such a manner that the described identity/trigger site pattern for the transmitter and the complementary recognition/target site switch composition has to be established. The most important requirement is to is to optimize these modules that the transmitter is only able to switches specifically, meaning only in the presence of both, identity AND trigger site.
Once the objective mentioned above is accomplished, the creation of an OR gate will be rather simple since it only requires two switches. However the creation of an AND or NOT gate and optimizing the logic gates to improve their responds function will remain the goal of future work. Also the creation of small networks and the correct integration of BioBricks as input and output molecules will be future challenges. Furthermore, we wanted to rather focus on the development and the testing of our structural design of the switches, rather than developing a variety of new BioBricks.

In silico design

As desribed above, our switches are based on certain design rules. However, there still are different strucutral paramters that need to be tested and optimized (length of recognition site and target site, choice of terminator, etc.). We used in silico design and modeling ) to test different parameters. Furthermore we tried to use the antitermination principle observed in nature, such as attenuation in E. coli or tiny abortive RNA´s of T7-phage.

Evaluation and Measurements

To evaluate the functionality of our molecular switches, we first had to establish several assays. Therefore, we improved an existing in vivo assay and developed an in vitro assay for this purpose. For more information please refer to the lab section.

Summarizing, the 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 transmitter/switch interaction reaction is 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 )

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: RFP or mCherry to measure the amount of produced output and eGFP for normalization. Our first 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 (compare Screening system: Backbone BBa_K494001). 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 very low basal transcription. A major advantage for the screening system. This newly designed screening approach renders the characterization of PoPS-based devices in general and switches in particular easy and robust. The low basal transcription furthermore fulfills one of the most important requirements for the designed switches, since output transmitters may only be produced in presence of an input transmitter. This helps to avoid strong "background" noise, which would extremely harden the successful interconnection of several switches.

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 switches after designing the screening system. Relying on the functionality of systems occurring in nature, we choose the HisTerm as well as the TrpTerm. Both switches are based on known natural [[2]]. Testing synthetic and none-naturally switchable terminators in vivo are goals for future work. Delorme et al. reported the His-Terminator to be a remarkable effective Terminator with more than 99% termination efficiency.^[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 transmitter, although the Modeling highly suggested the necessarily of finding an optimized transmitter lenght. Thus, the results are insufficient either to prove or 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 efficiency, 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, due to the mentioned required "yes or no" function (see Implementation and how to connect Biobricks). 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. Under circumstances, termination efficiencies altered by the transmitter are on a low range and not resolvable within observed 40% basal transcription.

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 characterization of our 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

Beside optimization of the reporter proteins in use, the major problem occuring in the experiments was the low capacity of the kit. The signal intensity was very low, which made it difficult to observe any signal intensity alterations, so no conclusion could be drawn from these measurements.

in vitro transcription

We used two completely independent in vitro systems: Using E.coli RNA Polymerase we analyzed the His and Trp switches that had already been tested in vivo. In a second set-up, we used the well-established T7 RNA Polymerase and switch based on the T7 terminator as well as several signal sequences.

T7 System

In contradiction to the results of Kang and coworkers and other groups, in our in vitro set-up the T7 terminator did not seem to terminate at all. The negative control (Promoter_Terminator_malachite binding aptamer) showed a similar increase in fluorescence as the positive control (Promoter_random sequence_malachite binding aptamer). Furthermore denaturing Polyacrylamide Gel Electrophoresis (PAGE) confirmed that there was no observeable termination of transcription. The addition of a signaling sequence led to a significantly lower increase in fluorescence, which can be attributed to the fact that both DNA sequences, switch and signal, compete for RNA Polymerases. However, there is almost no difference between the designed signals and random sequences, which is not a big surprise since there can be no antitermination if the terminator itself does not work.

Possible explanations for the contradiction between our results and those of Kang and coworkers might be the experimental set-up and the RNA Polymerases we used. Different variants of T7 RNA Polymerase might respond in different ways to terminator structures, and the termination might be influenced by the presence or absence of cofactors, depending on the purification methods used in producing the Polymerase.

This set-up offers a lot of possible experiments for the future, which we would have loved to conduct with a just a bit more time...

E.coli System

Compared to the T7 System, the E. coli RPO system produced poor increases in fluorescence, indicating little RNA synthesis. It was shown that the presence of a terminator decreases, as expected, the production of downstream RNA. This result was also confirmed by denaturing PAGE. However, due to the poor changes in fluorescence we were not able to actually characterize the behaviour of our switches in vitro, and the small RNA concentrations did not allow a quantitative interpretation of our gels. A major problem with this method was the low concentration of the ordered Polymerase resulting in a much weaker overall signal as comparable measurements using the T7 Polymerase.

In future experiments we might try to work with smaller volumes in order to reach higher concentration of RPO and of the synthesized RNA molecules, so measuring in 96 well plate readers might be a good choice.