RNA-based Delay: Biosynthetic AND gate

It has been proved that in transcriptional cascades the responses in time can vary among individual cells in a population, the loss of synchronization suggests the need for additional regulation devices [4] such as autoregulatory motifs or feed-back loops. In our project we decided to apply an extra control based on RNA and post-transcriptional control.

It has been shown how RNA molecules play important roles in regulatory mechanisms. Nowadays, synthetic biologists have elucidated many features of these molecules and have re-engineered them in order to increase or give new biological functions [6]. Besides, the modularity present in different biological circuits and logic gates analogous to those from electric components has allowed building biological components. One example of this, and in which we are interested, is biological AND gates. In this kind of components, the output depends on the presence of two inputs [7]. Riboregulators are RNA molecules which can perform both features, expression control and AND gate configurations. Therefore, we plan to construct a delay-device based on riboregulators (figure 9).

Figure 9. Biosynthetic AND gate. Combining an AND gate configuration and RNA-based post-transcriptional regulation, a device which might allows us to produce a delay is shown. Only when the key and the lock + ribosome binding site + GFP gene are together the output can be generated.

Preliminary approaches

Riboregulator

In bacteria, several factors affect translation initiation, including ribosomal recognition of the mRNA’s RBS and the start codon (AUG). Recognizing the importance of RNA interactions between the ribosome and RBS, and based on work on endogenous riboregulators, Isaacs et al. [8] sought to regulate bacterial gene expression by interfering with ribosomal docking at the RBS. From the outset, their objective was to create a modular post transcriptional regulation system that could be integrated into biological networks and implemented with any promoter or gene.... more.

Small interfering RNA

This approach is based on the gene silencing by small interfering RNA [9]. Protein 1 is always produced in the cell and is controlled by the constitutive promoter on gene 2. This protein must be selected carefully since the gene 1 producing the siRNA must be decided based on this protein and the siRNA must be specific for silencing the mRNA of protein 1 without disturbing the other mRNAs of the host organism.... more.

RNA-based Delay, Biosynthetic AND gate: The plan

Due to the lack of information on siRNA, the enormous advantage and information of riboregulators and the previous works of Isaacs F. 2004, Berkeley iGEM 2006, Caltech iGEM 2007 and Peking iGEM 2007, the riboregulator approach were selected. The construction of the plasmids is showed.... more.

Results

Main results

• Successful assembly of parts K175023 and K175024 which correspond to Plasmid 1 and 2 of the Biosynthetic AND gate section respectively.
• Electroporation of plasmid 1 and 2 into Escherichia coli TOP10 cells... more

See also Lock/Key library

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