Making it Rain

Playing rock-paper-scissors game with E.coli during summer was fun, but even if humans won, 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). （優子と相談する）

1.1 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), and that photo-oxidized isoprene acts as a condensation nucleus [4], and can cause shower(make it rain) even if it si present in very low concentrations.

It is known that the enzyme isoprene synthase is needed to catalyze the change of dimethylallyl-pyrophosphate(DMAPP) into 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) has isolated from the tree poplar (Barbara Miller et al., 2001). E. coli introduce this gene released isoprene into the air by diffusion [1]

In this study, we tried to make E.coli synthetize isoprene by the isoprene synthase on the standardized plasmid. Moreover we calculate that E.coli could produce isoprene more effectively and faster than the trees in the tropical rainforests. It means that the amount of isoprene produced by our E. coli is enough to form the secondary organic aerosols (and make it rain, but we still have to confirm the rain part).

1.2 Result

We constructed RBS-ispS as negative control and placIQ-RBS-ispS as positive control, using the placIQ promoter (BBa_I14032) and ispS. Gene ispS is extracted from the pMK backbone vector. (see more about our constructions)

We also measured the amount of isoprene from E.coli by Gas Chromotrography-Mass Spectrometry (GC-MS). When using GC-MS, we injected a series of chloroform-diluted liquid isoprene to draw the calibration curve. To confirm if liquid isoprene produced by E. coli would be released as a gas, we diluted liquid isoprene in water and also in LB medium. In both cases, we could confirm evaporated into the air (see more about these experiments).

Unfortunately, the GC-MS instrument got broken just before the wiki freeze. Therefore we were not able to conclude our experiments and report assay results. But we are certainly able to use the GC-MS again and report our results soon.

Discussion

計算結果 Note that both the negative control and the positive control produce amounts of isoprene greater than that required to form the aerosols when cultivated in 100ml of LB media (0.92[?g] and 9.6[?g], respectively), we can therefore conclude that, at least in principle, it is possible to make an E. coli that can make induce the formation of these secondary organic aerosols.

Isoprene by E.coli

Fig.3 Expected isoprene production by LT21 strain negative control pSB3K-placIQ and positive control pSB3K-placIQ-ispS is shown by blue columns respectively. 50ml sample of headspace gas from a 100ml LB culture which was induced with IPTG for 5 hours (Zao Y et al., 2011). On the right red column, the amount of isoprene needed to form aerosols is shown. The amount of isoprene required to form isoprene is 0.22-1.8[?g] in 400ml headspace (Tadeusz E et al., 2007)