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Team:SJTU-BioX-Shanghai/Background
From 2009.igem.org
Project introduction. Inspired by the natural regulator of circadian bioclock exhibited in most eukaryotic organisms, our team has designed an E.coli-based genetic network with the toxin-antitoxin system so that the bacterium oscillates between two states of dormancy and activity (more...)
Idea & Background
In this circuit, we used three key genes. The first one is relE. It is a toxin gene that terminates protein translation in that its product protein binds and cleaves the stop codon of an mRNA, thus prevents the mRNA from completion of translation, leaving an aberrant peptide and the ribosome occupied. When the amount of relE reaches a high level in a cell, the metabolism is suspended for lack of free ribosome, leading bacteria to domancy. RelE in bacteria is usually used to slow down the growth to protect the cell from starvation.
In order to 'wake up' the cell, a second gene, relB, is introduced as an antitoxin gene that dimerize with free relE in the cell.The relB proteins form a heterotetrameric (relB-relE)2 structure when binding with relE, which is too large to fit into the A site, so the toxic relE can be neutralized. Nevertheless, the effect of relE cannot be completely cleared up because the ribosomal A site can't be emptied by relB. So we introduced tmRNA, another key part in rescuing bacteria activity. It is a fascinating fact that tmRNA is a kind of RNA with the function of both tRNA and mRNA because it owns tRNA structure as well as mRNA sequence and an Ala in the 3' end. tmRNA rescues the stalled ribosomes through a process named trans-translation. It recognizes the ribosome with a nonstop mRNA and works through the procedure of accommodation and transpeptidation to release the mRNA. Then, the mRNA part of tmRNA functions as the template in translation so that the translation can be completed, resulting in a tagged protein that will be degraded by clpXP which most of the bacteria holds.
It is quite interesting to know that the relE cleavage rate varies with the stop codons as UAG>UAA>UGA, which means relE preferentailly cleaves stop codon UAG.So we modified the stop codons of relE, Lon and relB according to relE cleavage rate, with UAA, UAG and UGA respectively.
Once both the relE and relB pathways are unblocked, relE and lon are produced continually for some time whilst the relB proteins are hydrolysed by lon. At this time, the cell 'falls asleep' and remains in the state of dormancy. After while, the quantity of relE increases to a certain level so that production of Lon ceases, leading the amount of relB going up and waking the cell. What happens next is that since the majority of relE form dimer with relB, it loses the function of cleaving Lon's stop codons, resulting in the rise of Lon amount...
In this way the system oscillates between the states of dormancy and activation and may have the functions as a bioclock.
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