Part:BBa_K1230000:Experience

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We were able to characterise this part, since we found a measurable phenotype (kanamycin resistance). We also characterised it using Kanamycin �Minimum Inhibitory Concentration� (MIC) finding a MIC with MarA overexpression: 5 µg/ml and a wild-type MIC: 1 µg/ml A test was performed with three different antibiotics and different IPTG concentrations. The part seems to make the host sensible to Tetracyclin (maybe because it's on Cm plasmid, and both antibiotics act on similar ways), it seems not to have an effect on Ampicilin and it shows a relevant effect with Kanamycin. Which is further explored in the next figure.

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UNIQaa6d82ec13a1ded4-partinfo-00000002-QINU

Nagahama_2015

UNIQaa6d82ec13a1ded4-partinfo-00000003-QINU

••• iGEM_Nagahama_2015

We improved the characterization of a previously existing BioBrick Part BBa_K1230000 and submitted this improved BioBrick marA device as BBa_K1653006 to iGEM Registry. In exsisting part's information of marA, it gives E. coli resistance against kanamycin only. In this year, we confilmed that overepressing of marA gives E. coli resistance against geraniol as one of the terpene and decrease its intracellular concentration. This information is very beneficial for other iGEMers to production of organic substance that have toxicity using bacteria. The activator of AcrAB-TolC multidrug efflux pump exports some terpenes Export geraniol Fig. 1: Intracellular geraniol concentrations of E. coli JM109 (WT) and its overexpressing of marA strain, E. coli JM109 (marA). In this figure, intracellular content of geraniol was less in the strain E. coli JM109 (marA) than the strain E. coli JM109 (WT). The concentrations of intracellular geraniol from E. coli JM109 (marA) was 42.9 μg/ml, which was 40% lower than that from of E. coli JM109 (WT), 72.2 μg/ml. This figure is suggesting that internalized geraniol could be more efficiently exported through AcrAB-TolC efflux pump following the presumed activation of this gene by introducing the activator marA gene. The following graphs show that overexpressing marA gives resistance against geraniol and decrease intracelluler geraniol concentration. marA device BBa_K16530020 Resistance Fig. 2: Colony formation efficiencies of E. coli JM109 engineered with marA on geraniol overlaid plates. E. coli JM109 and E. coli JM109 (marA) were spotted on LBGMg agar plates in serial ten-fold dilutions (10⁻¹～10⁻⁵), overlaid with 1.0 % (V/V)　geraniol hexane solution (geraniol solution), and incubated at 30°C for 24 h. This figure shows that E. coli JM109 (marA) cells that overexpress the marA product is more survived on 1.0 % geraniol solution overlay plates than the counterpart control E. coli JM109 wild type cells. Fig. 3: Comparison of colony numbers after addition of 0.5 %( V/V) geraniol hexane solution (geraniol solution). Time interval for treatment was set every 1 hour from 1 hour to 4 hours. A: E. coli JM109 (WT) + hexane; B: E. coli JM109 (marA) + hexane; C: E. coli JM109 (WT) + 0.5 % geraniol solution; D: E. coli JM109 (marA) + 0.5 % geraniol solution. As shown in Figs. 2 A and B, treatment with hexane of E. coli JM109 (WT) and of E. coli JM109 (marA) showed similar colony numbers during these treatment intervals to those of time zero. This result suggests that hexane at this concentration and duration of time for 4hours did not affect both cell growth. In contrast, treatment with geraniol of E. coli JM109 (WT) and of E. coli JM109 (marA) showed toxicities to both strains (Figs. 3 B, C and D). If we watch the colony numbers carefully, E. coli JM109 (marA) had more than E. coli JM109 (WT) during these treatment intervals ((Figs. 3 C and D). These results demonstrate that toxicity of the geraniol was less to the strain E. coli JM109 (marA) than the strain E. coli JM109 (WT). Nagahama_2016 UNIQaa6d82ec13a1ded4-partinfo-00000005-QINU • iGEM_Nagahama_2016 We improved the characterization of a previously existing BioBrick Part BBa_K1230000 and (https://parts.igem.org/Part:BBa_K1230000)　the characterization of a previously existing BioBrick marAgene as BBa_K1653006 . In existing part's information of marA, it gives E. coli resistance against kanamycin and give E-Coli resistance against geraniol as one of the terpene and decrease its intracellular concentration. In this year, we confirmed that overexpressing of marA gives E. coli resistance against farnesol as one of the terpene. This information is very beneficial for other iGEMers to production of toxic organic substances that are produced using bacteria. We introduce an activator gene of AcrAB-TolC efflux pump (MarA) to release the farnesol from the cells and increase the content in the media that shows increase these flavors in the "Flavolator". In our study, we confirmed that overexpressing of marA gives host E. coli high resistance against farnesol. The activator of AcrAB-TolC multidrug efflux pump exports some terpenes Resistance Fig.7:Colony formation of E.coli JM109 engineered with marA on farnesol overlaid plates. E.coli JM109 and E.coli JM109 (marA) were dropped on LBGMg agar plates in serial ten-fold dilutions (10⁻¹～〖10〗^(-6)), overlaid with 30 % (v/v)　farnesol hexane solution (farnesol solution) and incubated at 30°C for 24 h. This figure shows that E.coli JM109 (marA) cells that overexpress the marA product better than the control E.coli JM109 wild type cells survived plates overloyed by 30 % farnesol solution. ; UNIQaa6d82ec13a1ded4-partinfo-00000007-QINU