You will be presented with the abstract, introduction, and discussion section from a biomedical research article. Your task is to create a summary that a layperson can understand, capturing the essence of the research article. Keep essential scientific terms, but ensure that the language remains clear and the concepts are explained in an uncomplicated manner.

Title: Binary addition in a living cell based on riboregulation}
Article:
Abstract:
Synthetic biology aims at( re-) programming living cells like computers to perform new functions for a variety of applications. Initial work rested on transcription factors, but regulatory RNAs have recently gained much attention due to their high programmability. However, functional circuits mainly implemented with regulatory RNAs are quite limited. Here, we report the engineering of a fundamental arithmetic logic unit based on de novo riboregulation to sum two bits of information encoded in molecular concentrations. Our designer circuit robustly performs the intended computation in a living cell encoding the result as fluorescence amplitudes. The whole system exploits post-transcriptional control to switch on tightly silenced genes with small RNAs, together with allosteric transcription factors to sense the molecular signals. This important result demonstrates that regulatory RNAs can be key players in synthetic biology, and it paves the way for engineering more complex RNA-based biocomputers using this designer circuit as a building block.
Introduction:
In 1945, von Neumann established the foundations of the logic architectures behind computers in his famous “First draft of a report on the EDVAC”[1]. There, the arithmetic logic unit( ALU) appeared as a principal element in the central processing unit. An ALU is a digital-like circuit that performs arithmetic and logic operations over bits of information. Certainly, today’s computers mount complex ALUs to deal with large volumes of information[2]. But in an emerging scenario of unconventional modes of computation[3], we could wonder whether ALUs, even if in simple forms, are implementable by other means. In particular, is it possible to engineer genetically such a device in a single living cell? Importantly, this question had a positive answer with the engineering of a genetic half adder in mammalian cells[4], by combining transcription factors( TFs) and RNA-binding proteins. A half adder is a basic implementation of an ALU to perform the binary sum of two bits of information. This requires generating two output channels, one for the sum( multiple of 1) and another for the carry( multiple of 2). Later, a genetic half adder was also engineered in bacterial cells exploiting combinatorial transcriptional regulation[5]. However, these designs are centered on regulatory proteins, which are limited in number, especially those with high propensity for composability and orthogonality with the host machinery, and do not allow an easy computational design of de novo sequences. In this regard, and even though ground-breaking work is being accomplished on circuit design automation[6], directed evolution of TFs[7], and de novo protein design[8], alternatives to protein-based regulation are required. In recent years, RNA has been exploited as an ideal substrate to engineer gene expression programs that robustly run in vivo, thanks to its functional versatility[9, 10] and model-based designability at the nucleotide level[11, 12]. Examples of this suitability are novel mechanisms of gene expression control through the modulation of transcription with non-coding RNAs[13–15], or chimeric RNA molecules integrating different domains that are able to transduce molecular signals[16–18]. Moreover, efforts in RNA synthetic biology to increase the sophistication of the designer systems have led to combinatorial logic gates[19], serial cascades[20, 21], a feed-forward loop[22], and a pulse counter[23]. In this work, we go one step further with the engineering of a genetic half adder in Escherichia coli centered on regulatory RNAs. In particular, we focused on riboregulators of translation initiation[24, 25] to implement our design. The whole system also relies on TF-mediated regulation, especially to sense the molecular signals and express accordingly those riboregulators. Interestingly, a genetic half adder would allow mounting a common response against two different molecules acting individually( mediated by the sum), and mounting a new response when they act together( mediated by the carry). This would be useful, for instance, in scenarios in which there is synergy between molecules[26].
Discussion:
We have programmed a bacterial cell so that it can perform the binary sum of two bits of information, encoded into the concentrations of IPTG and aTc( signal molecules). The bacterial cell reports the sum of this computation as red fluorescence and the carry as green fluorescence, a sort of minimal biocomputer. To achieve this dynamic behavior, we engineered a genetic system exploiting riboregulation[24]. The whole system consists of four synthetic riboregulators( RAJ11, RAJ11min, RAJ21, and RAJ12), three TFs( LacI, TetR, and cI), and two fluorescent proteins( mRFP1 and sfGFP), which work together within the cell in an articulate manner. Such a system did not require fine-tuning promoters or RBSs to perform as designed, in contrast to what might happen in other cases[43, 44]. Moreover, one important advantage of our designer circuit over the two previous genetic half adders[4, 5] is that the reporter gene conveying the sum is not duplicated. This makes the architecture to be better organized and more scalable, as already pointed out[1]. In addition, the genetic footprint of our designer circuit was greatly reduced thanks to the use of regulatory RNAs, with respect to circuits fully implemented with TFs[6]. The RAJ11, RAJ12, and RAJ21 sRNAs are of 55–71 nucleotides( excluding the terminators), and the RAJ11min sRNA is even of 30 nucleotides. Certainly, the DNA sequence required to encode a protein of average size is much longer. The cis-regulating regions at the DNA or RNA levels, by contrast, are of similar size. The PLlac and PLtet promoters are of 54 nucleotides and the 5’ UTRs involved in riboregulation of 52 nucleotides. Beyond this, by only mutating the seed region between the sRNA and the 5’ UTR it is possible to create riboregulatory systems that perform orthogonally in vivo[45]. This way, we might easily scale up our designer circuit. Following this strategy, of note, we already created a RAJ11-derived orthogonal system[36]. Definitely, we chose a given molecular implementation, but other implementations might be possible maintaining the same regulatory architecture. As the system does not rely on combinatorial promoters, nothing prevents the use of other input signals( e. g., endogenous substances of the cell) to perform the computation replacing the PLlac and PLtet promoters by suitable responsive promoters[43, 44]. Alternatively, LacI and TetR might be computationally redesigned to sense new compounds[46]. The riboregulatory mode, here characterizing an internal layer of gene expression activation, is also flexible. Cis-repression of translation might occur by trapping the start codon, instead of the RBS, in the 5’ UTR structure[25]. More distinctly, the activation might be transcriptional with sRNAs that act in trans as anti-terminators[14]. In addition to LacI and TetR( working in the sensory layer), our system also involves the TF cI to implement an internal repression in the XOR logic gate. We tried to implement this repression by antisense RNA[47] or CRISPR interference[15], without successful results( section C in S1 Appendix); arguably, because the expression of mRFP1 was from a high-copy plasmid. This reveals the necessity of pursuing the development of novel RNA-based mechanisms and circuits. All in all, our genetic implementation of an ALU promises to be important in the future to develop smart cells( e. g., diagnostic bacteria for clinical use) that can make appropriate decisions after certain processing( computation) of the signals perceived from the medium[48].
In this work , we have engineered a distinctive genetic system , based on regulatory RNAs that control the process of protein translation , that is able to perform arithmetic logic computations ( additions ) in a single bacterial cell . The system expresses as output fluorescent proteins according to the molecular concentrations of the inputs ( binary code ) . In the future , this circuitry might be instrumental to develop smart bacterial cells that can make appropriate decisions after certain computation for biomedical applications .