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From 2014.igem.org

RNAT·DESIGN

Why we introduce temperature control-RNAT (RNA Thermometer) to our IgEM?

Temperature is essential to the attachment and penetration process of M13 phage.

The character of M13 phage has been introduced particularly in M13(加链接) part. Here, we want to highlight the effect of temperature to the attachment and penetration process of M13 phage.

In 1964, Helen Tzagoloff and David Pratt found that the attachment rate of M13 phage depends on the temperature over the range from 0℃ to 45℃, and the penetration process of attached M13 phage is also affected by temperature dramatically【1】. The figures from 1 to 3 showed the results of the experiment done by them. At 0℃, phage attachment was allowed to take place, however, no detectable penetration would take place. When the temperature raises, for example, at 30℃ and 37℃, penetration of the attached phages can be easily measured, and the penetration rate increase dramatically between 15℃ and 37℃.Also, from 0℃ to 25℃, the attachment rate increase 2-fold, and remains the level to 37℃.

Inspired by this nature of M13 temperature-dependent penetration process, we suggest that if we set the primary infection temperature low to 15℃, even to 0℃, the penetration process of attached M13 phage could be synchronized, and then, for making our device more controllable, orthonormal and engineered, we could also achieve the synchronization of the release of M13 offspring via such temperature control. So, in our IgEM, we introduced a temperature control part-RNAT(RNA thermometer) to the M13 plasmid to regulate the expression of pⅢ, an essential protein related to the release of M13 offspring.

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RNAT is a structured cis-regulatory element responding to temperature changes in an immediate manner on the translation level.

In the field of synthetic biology, there are several strategies to realize temperature control, including using temperature-sensing promoters and introducing RNAT as 5’ UTR of mRNA of the target protein. Considering that the bacterial two-hybrid system (B2H)(加链接) we used regulates the expression of PⅢ on the transcription level, so the promoter cannot be adjusted optionally, and there is inevitable leaked expression of the background in B2H system, we need a regulation occurring on the translation level.

RNATs(RNA thermometers) are widely known to regulate many prokaryotic genes encoding heat shock proteins and virulence factors in their mRNAs[] and they act as riboregulators mediating temperature responsive regulation of these downstream gene by modulating the accessibility of its RBS, which affects efficiency of translation initiation[].when temperature reaches to the threshold value(resembling the chemical signal to riboswitches), the complex secondary RNA structures comprised by RNATs undergo a conformational change. RNATs also have the following advantages: 1) RNATs shows a rapid and cost-effective response to temperature changes as they control the translation of nascent or already existing mRNAs; 2) RNATs can detect temperature variations on the 1 °C scale as precisely calibrated sensory devices[]; 3)the closed conformation are tightly stable under the threshold. Thereby, RNATs can serve as an ideal temperature-controlled switch in our IgEM.

Functional theory of RNAT

At low temperatures, the 5ʹ untranslated region of the mRNA of these genes folds into a structure, which traps the ribosome binding site (RBS), or, the Shine-Dalgarno sequence (SD sequence), and then blocks ribosome access. As the temperature gradually raises, the equilibrium between the closed(OFF) and open(ON) conformations towards the open structure is shifted in a zipper-like manner, leading to the liberation of the SD sequence, thus permits formation of the translation initiation complex and increasing the efficiency of translation initiation.[]

RNAT in our Integrated Evolution Machine (IgEM)

In our design, synchronization of M13 phages’ behavior should be maintained by temperature control through the whole evolution process. Thereby, we plan to set the initial temperature of our IgEM to 0℃, M13 phages only attach to E. coli. And then, we raise the temperature to 30℃, M13 phages penetrate the M13 plasmids, and undergo diversification and selection via B2H system and mutation module respectively , but the translation of pⅢ is inhibited. When the host cell is ready, we finally raise the temperature to 37℃ for pⅢ translation, and with the expression of pⅢ, the M13 phage offspring can be released, so that to complete the amplification step.

To achieve the temperature-controlled expression of PⅢ, we need to add the DNA sequence of one typical zipper-like RNAT between the promoter used in B2H system and the coding sequence of pⅢ in the M13 plasmid, as the 5’ UTR of pⅢ mRNA after transcription. And when the temperature raise to or over the threshold temperature, PⅢ could be translated and M13 phage offspring can be generated and released for next infection.