• Background
• Methods
• Results
• Discussion
• Future work

FUTURE WORK

Owing to the pressing time and the lacking of ribosome binding sites (RBS) of different efficiency, we cannot get enough data to achieve our goal of regulating RBSs. However, we were illuminated by the project did by Peking University in 2011 [1]. We get 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 conform the RBSs’ sequence of 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 showed 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 stopped. So TPP ribozyme 2.5 is a statistical ribosome binding sites 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 owing to its complicated operations. So we need to change them into simple 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. They are all the same but some delicate factors [2].

Therefore, we will employ K598005 into the work we have done. As the work we have did the year, we will regulate the translation of leucine dehydrogenase (LeuDH). And the method is that we will use K598005 as a RBS of LeuDH. Then, we would change the concentration of TPP, and find the best concentration of TPP. Finally, we will get the RBS sequences with designed strengths by means of RBS calculators, which will be synthesized and employed in the final circuit. Then, this work is improved and finished.

Then, we will employ this method into other cofactor regeneration systems.

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)