Boolean Gates Based On Translational Controls

Boolean gates are normally used in electronics.These devices work on binary signals that take only two values: 0 and 1.Basic Boolean gates accept one or two input signals and return a single output signal.Boolean gates are also known as logic gates.They were named after 19 th century mathematician George Boole,who worked on algebraic system of logic.Boolean gates in biology allow gating of populations of cells based on ‘AND',‘OR' and ‘NOT' logic.This can be useful for finding cell populations that express antigen a and b,but not c.Boolean gates are useful when sorting cells stained with multiple fluorescent probes.For example,if you have a complicated hierarchical gating scheme,it can be useful to add in a ‘NOT' gate.This is to make sure that you are sorting only the cells that are of interest to you.You can also add Boolean gating to your hierarchical gates when looking for rare sub-populations,stained with multiple dyes.Building Boolean gates using DNA sequences is not a simple task.However,biological Boolean gates can be used,for instance,to carry out DNA-based computation or to construct biosensors.Hence,many ongoing research works in Synthetic Biology are finalized to the implementation of Boolean gates in vivo.This work aims at testing methods,based on RNA interference,for the construction of Boolean gates in the yeast Saccharomyces cerevisiae.In particular,we focused our attention on the so-called NOT gate that returns 1 when its only input is 0 and vice-versa.For this reason,NOT gates are also referred to as inverters.In biology,0 and 1 correspond to low and high concentrations of molecules(gate input)or fluorescence level(output).In our work,logic behavior is due to RNA interference(RNAi).RNAi is the acronym that refers to the RNA interference.This is a type of system occurring within living cells and plays a vital role in the control of active genes.It also helps determine and influences how these genes are actively working.The core of the RNA interface refers to two different types of smaller types of RNA molecules.These are the mi RNA or also known as the micro RNA.The other component is the si RNA or referred to as the small interfering RNA.Basically,RNAs are products of the genes and these could also bind with other genes such as m RNA.This could actually influence the activity of the compound whether it increases or decreases.One of the most salient roles of RNAi is for the defense of the cells especially in fighting off against certain parasitic genes.These are parasitic genes which are referred to as transposons and viruses.Most importantly,it is also necessary in the proper direction of the development of the cells and the general gene expression.This pathway is conserved among many eukaryotic species but is absent in the budding yeast S.cerevisiae.However,RNAi can be restored in S.cerevisiae by expressing the Dicer and the Argnaute protein from other budding yeast species,such as S.castellii and C.albicans.Here,we tested three main designs for RNAi-based NOT gates.They differ for the modality of si RNA precursor production.In our first implementation,the si RNA precursor is a long hairpin transcribed by a single promoter;in a different design,the si RNA precursor is produced by two convergent promoters.Finally,our third circuit uses two different promoters to synthesize an RNA molecule and its reverse complementary sequence.Each gate takes galactose as input and returns fluorescence as output.The presence of galactose induces the production of si RNA precursor;the si RNA precursor is processed by the Dicer into si RNA molecules that target the m RNA of the yeast enhanced green fluorescent protein(y EGFP).si RNAs bind the Argonaute and allow it to reach and cleave the m RNA of the y EGFP.Upon cleavage,the m RNA of the y EGFP is quickly degraded and fluorescence decreases.Hence,the circuit output is a low fluorescence level(0)only when galactose is present(1)in the medium where yeast cells grow.We managed to construct properly working NOT gates by using the convergent-promoter and the sense-antisense architecture.In contrast,Boolean behavior did not arise when a hairpin was used as a si RNA precursor.Further experiments are necessary to better understand how to make this particular gate design work too.According to Drinnenberg and co-authors,this is due to the fact that an antisense promoter,probably in the proximity of the locus where the plasmid is integrated,leads to the transcription of the si RNA precursor in glucose-containing media.As a first test to this hypothesis we placed the transcription unit that contains the hairpin into a centromeric plasmid.Then,we transformed the new vector into yeast.In principle,the centromeric plasmid should not contain any antisense yeast promoter.However,the final result did not change.We then asked the Genewiz company to synthesize a new plasmid where hairpin sequence is followed by two terminators: one in the sense direction(as usual)and one in the antisense orientation.The latter should prevent RNA polymerase II from transcribing the si RNA precursor after binding a promoter on the reverse DNA strand.Again,the synthesis of a DNA sequence containing a long hairpin turned out to be difficult even for a company and we did have not received this plasmid yet.In the future we plan to improve the efficiency of our working NOT gates,mainly by lowering the fluorescence level in presence of galactose.Moreover,more than a single type of molecules will be considered in order to trigger RNAi such that we will be able to build more complex Boolean gates.