Team:BIT-China/Questions and answers
Frequently asked questions:
Q: How can you control the regulation range of pH?A: The regulation range of pH is decided by three factors, the responsive range of pH-induced promoters, the efficiency of recombinase system and the strength of functional acid and alkali. The responsive range of pH-induced promoters is a rough range. Different efficiency of recombinase system combined with the responsive range of pH-induced promoters can result in various expression level of functional genes and thus we can get different amount of acid or alkali. Different functional acid has different pKa value. Considering all these three factors, we can control the regulation range of pH.
Q: Will the system function influence the bacteria normal division?
A: The gene in our system is harmless to our engineering bacteria. And most of our regulation products are endogenous, such as lactate, GABA and others. There may be another question that the acid we employed is from the citric acid cycle, so it might influence the growth of bacteria. But according to our experiment and some references, it has less effect on the cell division.
Q: Temperature regulation is also an important step in fermentation, have you ever considered the bad effects of heat which generated by neutralization if you use acid and alkali to regulate the pH?
A: Firstly, the neutralization process is not quick or violent here, especially the functional acid and alkali we used are medium. Therefore the heat generated by this process will dissipate quickly. Second, the growth of strains will also higher the temperature of fermentation liquor. Companies now already developed modern technology to keep the fermenter at a proper temperature. Last but not least, our project in 2013 is Intelligent Heat Regulation Engine which successfully solved the problems caused by heat. We can integrate this engine with pH Controller.
Q: How long does pH Controller takes to react with the environmental pH changes?
A: The reaction time mainly depends on the pH-responsive promoters. The reaction time of two promoters used in our basic circuits is half an hour, according to our experiments. More importantly, by error-prone PCR, we selected plenty of promoters which have different pH responsive range and various reaction time. Therefore, we can choose different promoters relying on different regulation needs. Besides, if the regulation circuits cannot quickly react to the changes, we still have resistance system which can maintain the homeostasis of bacteria. Thus the bacteria can have an enough time to sense the change and make regulation circuits function.
Q: Have you ever considered the issues related to biosafety? Do pH Controller need suicide device?
A: Yes we considered the concern for biosafety. Two central aspects of our project may cause biosafety concerns. The first is whether the acid and alkali produced by bacteria are harmful and the second is where the pH Controller will be ‘placed’. For the first concern, D-Lactate produced by ldhA is toxic for human. But we just use it for our basic verification and test the function of our design. Its use will depends on the real fermentation situation since D-Lactate is also an important product in fermentation. What’s more, we have already found functional genes which can produce harmless L-Lactate. Except D-Lactate, most of our functional acid and alkali we used is not harmful to human, and some of them are salutary for us human beings. As for the second concern, we can ‘place’ our pH Controller in engineering strains as well as another kind of strains for co-fermentation or immobilization. In other words, where the pH Controller function is decided by the fermentation situations and regulation needs. If it is used for co-fermentation, we may apply suicide device.