Team:BIT-China/Description
Nowadays, fermentation industry is crucial for a country’s economy. In China, fermentation industry output reached 2,000 billion RMB in 2011, and till 2013, this number has increased to 3,000 billion RMB (Fig.1). Fermentation industry’s output accounts for about 3 percentage of GDP in China, and it’s predicted that the output will reach 10,000 billion at the end of 2015. As a result,the optimization and improvement of fermentation process is important.
However, problems concerning fluctuation of pH in fermentation are usually knotty. Firstly, the activity of enzymes can be tremendously influenced by environmental pH. The activity of enzyme will plummet at an improper pH, and eventually the fermentation effectiveness will decrease. Worse still, a different pH can entirely change the microorganism’s fermentation products. For example, Aspergillus niger can produce citric acid at pH 2.0 ~ 3.0, while the product will be changed to oxalic acid at pH 6.5 ~ 7.5. As for corynebacterium glutamicum, the product is glutamate at pH 7.0 ~ 8.0 while at pH 5.0 ~ 5.8 the products are Glutamine and N-aceglutamide. Therefore, a suitable environmental pH is a key factor for higher fermentation productivity.
Fig.1 Fermentation industry output from 2005 to 2013[1].
To maintain fermentation pH range, people have found some methods to regulate the pH of fermentation liquor, such as probe detection technique combined with computer programs, and adding acid and alkali into fermenters straightly. However, environmental interference can strongly interrupt probe detection, and increase reaction time. What’s more, adding large amount of acid and alkali may cause unbalanced pH in different areas in fermenter. Those problems will decrease fermentation productivity and effectiveness. More importantly, all of these methods which heavily rely on electrical technology will increase factories' costs and energy consumption, but none of them considered the function of microorganisms themselves.
So, can we regulate environmental pH in a simpler way and meanwhile maintain the effectiveness?
Is there any possibility to regulate environmental pH by microorganisms themselves?
Based on these questions, we come up with our idea. Here is the bacteria, now it faces this serious problem: how to survive in acidic and alkaline environment, and regulate environment to a suitable pH, to protect the fermentation world?
At that moment, pH Controller appears. When the bacteria get this weapon, it can balance the pH to a suitable level. To achieve these goals, we firstly constructed PC in E.coli, and designed two subsystems in gene circuits. First system is resistance system to ensure survival of bacterium and regulate intracellular pH, while the second system--regulation system, focuses on regulating environmental pH to a suitable range.
Precise artificial pH control raises worldwide concern because fluctuation of pH level can tremendously influence the performance of cell. This year BIT-CHINA designed a micro-machine, pH Controller, which can make bacteria survive in both acidic and alkaline environment, and adjust the extracellular pH to an optimal level.
Our project consists of two components, basic circuits and fine-regulation circuits. Basic circuits contain the resistance system and the regulation system. The resistance system makes bacteria survive in an expanded range of pH. The regulation system adjusts the environmental pH by synthesis of acid and alkali. Fine-regulation circuits are proposed for different regulation requirements.
We established three libraries in fine-regulation circuits, one for the pH-responsive promoters generated by error-prone PCR, another for various inversion effectiveness recombinases and the one left is for functional genes producing acid or alkali. All these factors decide the regulation range of pH. Therefore, we can meet different regulation needs.
Our pH Controller allows for lower artificial pH regulation costs in industrial fermentation. The hosts with our pH Controller can not only maintain high fermentation efficiency but also fine regulate the microenvironmental pH.
This year, we improved one part BBa_K117003 by making it compatible with RFC [10] and then characterized the new part’s function. We also verified the function of two standard parts BBa_K137058 and BBa_K907004.
The activities of P-atp2 at different pH from 5.0 to 9.0 are gained by measuring the activity of enzyme β-galactosidase. Meanwhile, we moved the EcoRI site of the promoter by one day step mutation so that we could standardize the promoter P-atp2 and submit the standard part to iGEM (In fact we submitted P-atp2 +LacZ alpha BBa_K1675022).
At the same time, we verified the function of FimE and Bxb1 device through fluorescence respectively. We ligated the mRFP and K137058 together, aiming to evidently verify that the promoter pTetR has been inverted rather than be silenced. We chose pTetR as the promoter which could be induced by tetracycline in K907004. According to the fluorescence result, we can obviously find that the fluorescence verification device K137058 and K907004 worked as expected. Meanwhile we also proved the function of FimE and Bxb1 gf35.