iZJU-China

Introduction

Due to the volatility of α-pinene in turpentine, long-term exposure to such an environment will be very harmful to human health. Therefore, our project intends to solve this problem. As planned, our product will be put in places like art studios with the aim of degrading the detrimental chemical substance.

In the following context, a series of proofs will be presented to demonstrate the viability of our product from perspectives including testing the vitality of engineered bacteria, mimicking the working environment of engineered bacteria, and detecting the effect of the degrading process.

Preliminary Experiment

In our product, the engineering bacteria fixed in the biological filter need to work in an environment with high concentration of α-pinene. It has been proven that E. coli with P450BM-3 QM, GlcDH-II and GLF can oxidate α-pinene into α-pinene oxide (1). Therefore, we assume that this kind of engineering bacteria can live in the environment with both α-pinene or α-pinene oxide. We know that it is necessary to test the tolerance of this engineering bacteria to α-pinene. However, due to the difficulties we encountered in the co-transformation of two plasmids, we have not yet tested this engineering bacteria.

Another research demonstrated that E. coli with Prα-POL can degrade α-pinene oxide (2). Here we conducted Kirby-Bauer tests to see if this engineering bacteria can deal with high-concentration α-pinene or α-pinene oxide. We tested the growth of engineering bacteria introduced with prα-pol plasmids at 75% and 100% concentrations of α-pinene oxide. In the three repeated experiments, both two groups had inhibitory rings smaller than 3mm. The radius of inhibition rings of the two groups was close in each replication group. Therefore, we can conclude that this engineering bacteria have low sensitivity to high-concentration α-pinene oxide and are able to adapt to the high-concentration α-pinene oxide environment.

Figure 1. Kirby-Bauer tests of E. coli with Prα-POL

Two-phase culture system

In our product, we designed to fix the engineering bacteria on biological filter, which can be infiltrated with culture media to support the grow of bacteria. To simulate the situation, we resorted to the aqueous-organic two-phase culture.

Figure 2. The diagram of aqueous-organic two-phase system

Because of the difference in density, the layer of α-pinene (organic phase) was located above the layer of LB medium (aqueous phase) where the engineering bacteria with glf plasmids and p450bm-3qm-glcdh plasmids living. Rotated by a shaker, these two layers were thoroughly mixed, allowing bacteria to degrade α-pinene. Similar experiments were conducted to mimic the environment containing α-pinene oxides where engineering bacteria with prα-pol plasmids living. In addition, two engineering bacteria working together to degrade α-pinene were also simulated in an environment where α-pinene was the organic phase.

These experiments mimic the dynamic cultivating environment in our product. In addition, the increased contact area of the engineered bacteria and chemical substances, which can improve the degradation efficiency.

Degradation Effect

To test if our engineering bacteria can successfully degrade α-pinene, we used mass spectrometry (GC-MS) for detection. The effect of two kinds of engineering bacteria were tested separately. Figure 3 shows the degradation effect of α-pinene oxide by E. coli with Prα-POL. Results indicated that after 3.5 hours of biphasic culture, α-pinene oxide can be converted into isonovalal. Similar experiments will also be done to detect the degradation effect of α-pinene by E. coli with P450BM-3 QM and GlcDH-II.

Figure 3. The mass spectrum was generated for analysis. This revealed that both α-pinene oxides (A) and isonovaval (B) were detected in the GC-MS analysis.

Retrospect and Prospect