Although classical molecular biology and genetic engineering equipped the science community with many functional proteins and possible applications, using and linking different parts together to a working network still requires highly complicated strategies. The switches established in molecular biology, protein expression control units like the lac operon, are highly limited, as most of them rely on interactions including metabolites and/or proteins. Thus, it is hardly possible to build up a logic network inside a cell without interferences between different switches. Our approach is to change this by developing a new and more robust way to control E. coli cells using RNA-RNA-interaction based switches, which we call bioLOGICS.

A network consisting of AND, as well as OR circuits can be easily constructed with these switches. We designed our switches in a way, that makes upscaling no problem: This is a major advantage to ribozyme and especially protein based networks. While the complexicity of protein-protein interactions may work for cells, constructing networks without just copying complete operons is hardly possible. With the small size of our bioLOGICS, 10 logic units occupy the space of an average protein sequence on a plasmid. Circuits based on bioLOGICS may play a key role for gene regulation with more variations than just on/off in the future.

The idea behind our project is to change the way BioBricks have been used up to now. Over the years, many receptors and signals have been constructed as BioBricks during the annual iGEM competition, but still it is not possible to interconnect these Bricks in a complex biological network resuting in a cell, that is able to respond to its environment giving differenciated responses depending on the input signals. (Beispiel: cambridge hat das gemacht, xx dies, aber eine zelle kann nicht beides...
We plan to create biological switches, that can function as locial gates inside a cell. Our switches rely on RNA/RNA-interactions, regulating transcriptional termination. This is a major advance of our concept, as regular switches rely on complex regulation including proteins and/or metabolites. Thus, our switches shall offer a greater robustness and their behaviour should be easier to predict. Read more (hier sollte noch das hochskalieren erwähnt werden...
These switches can further be used to build up a logical network inside a bacterial cell, enabling every scientist to connect as many functionalities (in form of BioBricks) as designated. We plan to offer simulation on each specifically designed network.

Our visioon: A logic network inside the cell

The logic network we want to apply will be based on devices, that can be easily upscaled and therefor offer the chance to build networks of any wanted complexicity. Our devices rely on pure RNA/RNA interactions and thus their behaviour is well predictable.

The concept we rely on for our design of RNA-switches is based on the principle of attenuation.

Experiments

We designed several experiments to test our switches, all of them based on fluorescence measurements. We designed experiment setting for measurements in vivo as well as in vitro. Our in vitro measurements relied on two different experiment set-ups. While the first was based on a commercial E. coli-lysate, the latter was reporting on a transcriptional level only, eliminating most of the possible side-effects one could expect in the complex behaviour of a living cell or cell-lysate. Read more

Results

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thing to move

bioLOGICS: Logical RNA-Devices Enabling BioBrick-Network Formation Abstract Among the goals of iGEM is the creation of synthetic biological parts and their utilization to achieve novel features and behavior in biological systems. The emphasis of our project is put on this latter, "systems" aspect of iGEM. More precisely, we aim at the development and experimental demonstration of a scalable approach for the realization of logical functions in vivo.

By developing a computational biological network based on RNA logical devices we will offer everyone the opportunity to 'program' their own cells with individual AND/OR/NOT connections between BioBricks of their choice. Thereby, BioBricks can finally fulfill their original assignment as biological parts that can be connected in many different ways. We will achieve this by engineering simple and easy-to-handle switches based on predictable RNA/RNA-interactions regulating transcriptional termination. These switches represent a complete set of logical functions and are capable of forming arbitrarily complex networks.

The Experiments

Fluorescent proteins as reporter

Our initial idea to prove our concept of antitermination was to use flourescent proteins as reporters. This approach gives the opportunity to measure the termination and antitermination efficiency of our designed BioBricks in vivo as well as in vitro, the latter using a translation kit based on e.coli lysate.
We decided to use the flourescent proteins GFP and RFP, as their spectra do not overlap and we would not measure any FRET. We would use GFP fluorescence as internal control and RFP fluorescence as signal to detect termination/antitermination by our switch we cloned in between the coding sequences of the proteins. Both protein sequences are under the control of one (L-arabinose induced) promoter.

When measuring the termination of our BioBricks and the antitermination by their corresponding signal-RNA, we should be able to observe an increasing RFP emission compared to the GFP emission upon induced signal-RNA production in the cells/in the kit:

Wiith these measurements, it should also be possible to observe differences in efficiency of termination as well as antitermination between our designed switches.

Measurements with the malachite green aptamer as reporter

A second possibility to measure parameters of our switches we came up with, was the idea to investigate our system on the transcriptional level only. Therefore, we decided to use malachite green as reporter. Malachite green in a fluorescent dye, whose emission increasing dramaticly (about 3000 times) upon binding of a specific RNA-aptamer.

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To study the switches on the transcriptional level gives the advantage, that we would have less interferences and possible artefacts. Also, we are not sure how cellular mechanisms like degradation of RNases or interacting factors as well as molecular crowding influence our systems.

Emission spectra of malachite green; A: without signal-RNA, B: with signal-RNA

We made constructs comprising of a sigma(70)-binding promoter followed by a short nonsense sequence, the switches and the aptamer sequence.
Also we made constructs, where the transcription of the signal-RNA is under the control of a sigma(70) promoter. These two linear DNA-constructs, together with the e.coli RNA-polymerase and the right buffer conditions should represent an easy-to-handle measurement kit on the transcriptional level.

Results 

Flourescent proteins

Unfortunatly, we had to change the reporter construct two times during our experiments as several problems occured in our measurements:

First Try: based on the measurement plasmid pSB1A10

At the beginning, we decided to use the reporter plasmid pSB1A10 from the registry. It consists of the fluorescent proteins eGFP and mRFP1. Each sequence includes a ribosome binding site and a stop-codon; the two genes are divided by a cloning side including the BioBrick cleavage sites.

pSB1A10

In front of the eGFP sequence, the plasmid includes an arabinose-inducable promoter. The plasmid also contains an ampicilline resistence.

We cloned our switches into the cloning site of the measurement plasmid and used an empty cloning site as control; our signal-RNAs we cloned into the pSB1K3 vector, together with the BioBricks R0011 (Lac promoter) and B0014 (double terminator of transcription). Afterwards, we cut pSB1K3 with Aat2 and Pst1 and pSB1A10 with Nsi1 and Aat2 and ligated those fragments of each plasmid that contained our Bricks to get a Monsterplasmid.
We had to do so, as both plasmids contain the same ori mechanism. In addition, having both the switch and the signal RNA transcribed from the same plasmid gives us a high local concentration of the signal, once its transcription is induced.

We transformed BL21(DE3) cells with the plasmid. We set up cultures, induced the arabinose promoter and measured the GFP and mRFP1 excitation/emission spectra within time.

Unfortunatly, we were ot able to detect any RFP signal, not even in the positive control with no switch in between the GFP/RFP sequences.

From these experiments, we concluded, that the mRNA of the RFP variant used was instable and rapidly degraded by RNases, so the RFP was not synthesized in the cells. This was also the conclusion from XXX...

As solution to this, we decided to design a measurement plasmid ourselves:

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Second Try: A measurement plasmid of our own design

To design our own plasmid to overcome the problems that occurred in our first try gave us tghe possibility to overcome several other problems:

Third Try: One promoter for each protein

We decided to use the measuremnt plasmid we developed in our second try but to clone another L-arabinose induced promoter into the plasmid, in front of our switch followed by the mCherry sequence.

In this way, we still can use GFP fluorescence as internal control, because both protein transcription is under the control of a promoter of identical design.

Though we are still not able to tell exactly why our previous measurements did not work, but with this construct we measured the first time fluorescence of the mCherry protein in our positive control.

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