Team:Tokyo Tech/Projects/making-rain/index.htm


Tokyo Tech 2011

Making it Rain

Illust Playing RPS with E. coli during summer was fun, but, even if humans won, celebrations did not last long since we soon returned to complaining about the hot weather. As a prize for humans who win in our RPS game, we designed an E. coli that can make it rain, making the hot summer more fun and refreshing (let alone applications in agriculture).

1. Introduction

To make it rain we focus on the substance isoprene. It has been observed that trees in tropical rainforests contribute to the formation of photo-smog aerosol in the lower atmosphere by releasing isoprene (Paulson and Seinfeld, 1992). The photo-oxidized isoprene acts as a condensation nucleus [2], may cause rain even if it is present in very low concentrations.

Fig. 1 Isoprene photo-oxidation reaction

It is known that the enzyme isoprene synthase can catalyze the conversion of dimethylallyl diphosphate(DMAPP) to isoprene. DMAPP is normally synthesized by E. coli, so the only thing we need to make our bacteria synthetize isoprene is isoprene synthase. The isoprene synthase coding gene (ispS) is isolated from the tree poplar (Barbara Miller et al., 2001). E. coli introduced this gene released isoprene into the air by diffusion [1]

Fig. 2 Formation of isoprene is catalyzed by isoprene synthase

In this study, we made E. coli synthesize isoprene by introducing ispS.

2. Isoprene by E. coli

To measure the amount of isoprene produced by E. coli with the introduction of ispS, we constructed negative control PlacIQ and sample PlacIQ-RBS-ispS(BBa_K649303) , using the PlacIQ promoter and ispS. Gene ispS is extracted from the pMK backbone vector. (see more about our constructions)

Fig. 3 Constructions of PlacIQ and PlacIQ-RBS-ispS

We used Gas Chromotrography-Mass Spectrometry (GC-MS) to measure the amount of isoprene produced by E. coli. When using GC-MS, we firstly injected a series of chloroform-diluted liquid isoprene to draw the calibration curve. Then the peaks of negative control(PlacIQ) and sample(PlacIQ-RBS-ispS) were detected at the retention time at 1.1 min. This is same to the retention time of the peak of authentic material isoprene. Therefore, we concluded that our E.coli was producing isoprene as we expected.

According to the calibration curve and peak areas, we calculated the isoprene produced by our E. coli BL21 (DE3) with the introduction of ispS is about 4.1×10-5 mg/L, while negative control (PlacIQ) only produced one eighth of the sample. (see more about this work.)

(a) (b) isprene-graph
Fig. 4 isoprene detected by GC-MS (This work is done by Yuto Sugiuchi.)
(a)a-1:negative control(PlacIQ), a-2:sample(PlacIQ-RBS-ispS), a-3:authentic material
(b)The amount of isoprene detected in E. coli extract.

3. Discussion

The reaction between isoprene and ozone has been studied to examine physical and chemical characteristics of the secondary organic aerosol formed. Aerosols is suspension of solid particles or liquid droplets in gas. The most common aerosol in the atmosphere are clouds, which normally consist of suspensions of water droplets or ice particles of greater density, and can later cause rain. According to those information, we designed an easy indoor experiment of reaction between isoprene and ozone, and confirmed that isoprene can make aerosol.The ozone-isoprene reaction was carried out in teflon bags. To facilitate the reaction, ultraviolet radiation was used. 20 mins after the reaction started, formation of aerosol was confirmed as shown the photos below.

Isoprene - Isoprene +
aerosol2 aerosol1
Fig. 7 aerosol formation

The picture on the left shows that when isoprene was not present no aerosol was detected even when air, water and ozone were put together under reaction conditions. On the other hand, the picture on the right shows that when isoprene was used, it formed an aerosol (this became evident because the trajectory of the laser light was visible). see more about this work.

All in all, we confirmed that E. coli with the insertion of ispS synthesizes isoprene and that isoprene makes aerosol. So our E. coli will make it rain! We also thoroughly concerned about the safety that might come up with the using of isoprene, details can be seen here.


[1] Yaru zhao, et al., Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway, Appl Microbiol Biothechnol(2011) 90:1915-1922
[2] Leonardo Silva Santos, et al., Mimicking the atmospheric OH-radical-mediated photooxidation of isoprene: formation of cloud-condensation nuclei polyols monitored by electrospray ionization mass spectrometry, Rapid Communication in Mass Spectrometry, 2006

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