Team:SIS Korea/Design
Design
Methods of Promotion
A key characteristic of our PETase producing E.Coli bacteria that set it apart from previous attempts at a similar product was its promoter sequence; specifically, the light-induced expression system. We wanted to find a sequence that would allow for efficient secretion of PETase.
Initially, we thought of two different approaches to the promotion of our PETase producing sequence.
First was the light inducible expression system that we later chose as our preferred form of promotion. This system also called the pDawn system, included a sequence of an I9 promoter, YFI gene, a Fix J gene, a Fix K2 promoter, and a cI gene phage λ repressor that would prevent the translation of the PETase gene unless provoked by light. The I9 promoter leads to the translation of YF1 which phosphorylates and activates Fix J, which then allows for the RNA polymerase to bind to the Fix K2 promoter. This leads to the activation of the cI gene phage λ repressor that prevents the usage of the pR promoter which leads to the production of PETase. However, if hit with a light wave with a wavelength of 470nm, a photoreceptor will alter the YF1 to inactivate the Fix J, which allows for the usage of the pR promoter and the production of PETase. The light inducible expression system is useful primarily due to its ease of regulation. With the flick of a light switch, one can control the production of PETase. Compared to other promoters, which may require specific conditions like the constant addition of certain chemicals to function, this system has much fewer requirements.
The other option was the constitutive expression system which simply produced PETase nonstop with the use of a strong promoter which would ensure continued translation. On paper, it would sound great to have a never-ending production of PETase. But when we tested the system, we discovered multiple complications that would render the constitutive expression system virtually unusable/unsuitable. First, the rapid, unending production of PETase resulted in a large metabolic load on the bacteria and made it more difficult to sustain the population. Also, the bacteria ended up producing excessive amounts of PETase to the point where inclusion bodies were formed and the PETase could not be secreted properly. 37 °C was the optimal condition for both expression systems, and this was an issue also found occasionally in the light-inducible expression system, but in the case of the light-inducible expression system the issue could be remedied by regulating the temperature at 18 °C whereas in the constitutive expression system 18 °C completely halted enzyme production.
In the end, it was clear that the light-inducible expression system was the better choice; not only was it capable of regulating production easily, but it was also easier to maintain and it produced enzymes more effectively.