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Team:Tokyo Tech/Projects/making-rain/index.htm
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<li><a href="#intro">1.Introduction</a></li> | <li><a href="#intro">1.Introduction</a></li> | ||
<li><a href="#Res">2.Result</a></li> | <li><a href="#Res">2.Result</a></li> | ||
<li><a href="#discus">3.Discussion</a></li> | <li><a href="#discus">3.Discussion</a></li> | ||
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Revision as of 13:22, 4 October 2011
Making it Rain
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 [3], can cause shower (make it rain) even if it si present in very low concentrations.
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) 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.
2. Result
We constructed negative control RBS-ispS and sample placIQ-RBS-ispS, 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.
3.Discussion
According to a paper [1], E. coli BL21 (DE3) harboring isoprene synthase will accumulate around 94 [mg/batch-l] isoprene, while E. coli with negative control will produce very little amounts of isoprene about 9 [mg/l]. The following calculations show that E.coli make a precipitation is feasible. If we culture this E.coli in 2L media set in 24m3(3m×4m×2m) space, the concentration of isoprene is 7.8ppm (=21.9[µg/l]). This concentration is higher than the requirement 3ppm (=8.4µg/(air-l)]) to form secondary organic aerosols in a reaction chamber. We also designed an easy indoor experiment of isoprene to form aerosol through ozone-oxidization of isoprene inside a Teflon bag. Though we estimate that from 3ppm (=8.4 µg/l) isoprene in the Teflon bag likely to form aerosol. [4]
4.Reference
[1] Yaru zhao, et al. Biosynthesis of isoprene in Escherichia coli via methylerythritol phosphate (MEP) pathway, Appl Microbiol Biothechnol(2011) 90:1915-1922[2]Mattijs K. Julsing, et al. Functional analysis of genes involved in the biosynthesis of isoprene in Bacillus subtilis, Appl Microbiol Biothechnol(2007)75:1377-1384
[3]O`Dowd, C.D. Aalto, K.Hameri, M.Kulmala, and Thorsten Hoffmann. Atmospheric particles form organic Vapours, Nature,2002.
[4] Nadine M. Czoschke et al, Effect of acidic seed on biogenic secondary organic aerosol growth, Atmospheric Environment, 2003
