FUTURE WORK

Owing to the limited time and the lack of ribosome binding sites (RBS) of different efficiency, we cannot get enough data to achieve regulating RBSs. However, we were illuminated by the project did by Peking University in 2011 [1]. And we come up with the idea that we could use genetic rheostats as means of RBSs' regulators. And then, we could get the excellent RBSs' efficiency and use RBS calculators to edit the RBSs' sequence to achieve a suitable efficiency.

Genetic rheostats are ligands-responsive RNA devices. From the kits, we can get a thiamine pyrophosphate (TPP)-regulated hammerhead ribozyme. This part is numbered K598003, which is a TPP down-regulated hammerhead ribozyme 2.5 with native RBS (Figure 1, [1]).

Figure 1 Mechanism for TPP ribozyme 2.5 [1]

As shown in Figure 1, TPP ribozyme 2.5 has a self-cleavage of hammerhead domain. At the absent of thiamine pyrophosphate, these RNA devices would cleave themselves and expose the ribosome binding sites. Then the translation processes start. However, TPP ribozyme 2.5 would transform to a structure that hides self-cleavage of hammerhead domain. As a consequence, ribosome binding site remains covered and the translation process stops. So TPP ribozyme 2.5 is a statistical ribosome binding site with which we can regulate the translation initial rate by changing the concentration of thiamine pyrophosphate.

However, statistical ribosome binding sites cannot be applied in industrial production due to its complicated operations. So we need to change them into normal ribosome binding sites, which can be achieved by means of RBS calculators. There are two kinds of thermodynamics-based RBS calculators online. They are the RBS calculator and the UTR designer [2].

Therefore, we will apply TPP ribozyme 2.5 to the work we have done. As the work we have done the year, we will regulate the translation of leucine dehydrogenase (LeuDH). And the method is that we will use TPP ribozyme 2.5 as a RBS of leudh. What's more, we would change the concentration of TPP, and find the best concentration of TPP. Finally, we will get the RBS sequences with designed efficiency by means of RBS calculators, and this will be synthesized and employed in the final gene circuit.

Reference:

[1] https://2011.igem.org/Team:Peking_R/Project/RNAToolkit
[2] Reeve, B., Hargest, T., Gilbert, C. & Ellis T. Predicting translation initiation rates for designing synthetic biology. Mini Review Article. 2, 1-6 (2014)