Difference between revisions of "Part:BBa K1230000:Experience"
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===Applications of BBa_K1230000=== | ===Applications of BBa_K1230000=== | ||
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+ | ===User Reviews=== | ||
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+ | We were able to characterise this part, since we found a measurable phenotype (kanamycin resistance). | ||
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+ | 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 | ||
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+ | https://static.igem.org/mediawiki/igem.org/8/8b/U102_Fig2.png | ||
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+ | 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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+ | https://static.igem.org/mediawiki/igem.org/2/21/U101_Fig3.png | ||
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+ | ==Nagahama_2015== | ||
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+ | <partinfo>BBa_K1230000 AddReview 3</partinfo> | ||
+ | <I>iGEM_Nagahama_2015</I> | ||
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We improved the characterization of a previously existing BioBrick Part [https://parts.igem.org/Part:BBa_K1230000 BBa_K1230000] and submitted this improved BioBrick ''marA'' device as [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1653006 BBa_K1653006] to iGEM Registry. | We improved the characterization of a previously existing BioBrick Part [https://parts.igem.org/Part:BBa_K1230000 BBa_K1230000] and submitted this improved BioBrick ''marA'' device as [https://parts.igem.org/wiki/index.php?title=Part:BBa_K1653006 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. | 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. | ||
<br> | <br> | ||
<br> | <br> | ||
+ | [[File:MarA dev.png]] | ||
+ | <br> | ||
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'''The activator of AcrAB-TolC multidrug efflux pump exports some terpenes''' | '''The activator of AcrAB-TolC multidrug efflux pump exports some terpenes''' | ||
− | <br> | + | |
+ | <br> | ||
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+ | ====Export geraniol==== | ||
[[File:GC conc fig.png |650px|thumb|center|Fig. 1: Intracellular geraniol concentrations of ''E. coli'' JM109 (WT) and its overexpressing of ''marA'' strain, ''E. coli'' JM109 (''marA''). | [[File:GC conc fig.png |650px|thumb|center|Fig. 1: Intracellular geraniol concentrations of ''E. coli'' JM109 (WT) and its overexpressing of ''marA'' strain, ''E. coli'' JM109 (''marA''). | ||
<br> | <br> | ||
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.]] | 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.]] | ||
− | ===Resistance=== | + | <br> |
+ | The following graphs show that overexpressing marA gives resistance against geraniol and decrease intracelluler geraniol concentration. | ||
+ | <br>'''[https://parts.igem.org/Part:BBa_K1653020 ''marA'' device BBa_K16530020]''' | ||
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+ | ====Resistance==== | ||
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+ | [[File:MarA plate assay yoshiharu did.png|750px|thumb|center|Fig. 2: Colony formation efficiencies of ''E. coli'' JM109 engineered with ''marA'' on geraniol overlaid plates. | ||
+ | <br> | ||
+ | ''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. | ||
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]] | ]] | ||
− | [[File:MarA plate assay nishikawa last.png|650px|thumb|center|Fig. 3: Comparison of colony numbers after addition of 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) + geraniol; D: ''E. coli'' JM109 (''marA'') + geraniol. | + | [[File:MarA plate assay nishikawa last.png|650px|thumb|center|Fig. 3: Comparison of colony numbers after addition of 0.5 %( V/V) geraniol hexane solution (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. | + | <br> |
+ | 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). | ||
]] | ]] | ||
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− | == | + | ==Nagahama_2016== |
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+ | 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. | ||
+ | <br> | ||
+ | <br> | ||
+ | [[File:MarA dev.png]] | ||
+ | <br> | ||
− | + | '''The activator of AcrAB-TolC multidrug efflux pump exports some terpenes''' | |
+ | <br> | ||
+ | <br> | ||
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− | + | ====Resistance==== | |
− | + | ||
+ | [[File:E.colimarA result .jpg|800px|thumb|center|Fig.7:Colony formation of ''E.coli'' JM109 engineered with marA on farnesol overlaid plates. | ||
+ | <br> | ||
+ | |||
+ | ''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. | ||
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Latest revision as of 22:01, 19 October 2016
This experience page is provided so that any user may enter their experience using this part.
Please enter
how you used this part and how it worked out.
Applications of BBa_K1230000
User Reviews
UNIQ5a124a9e99451196-partinfo-00000000-QINU
No review score entered. Username |
We were able to characterise this part, since we found a measurable phenotype (kanamycin resistance).
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.
|
UNIQ5a124a9e99451196-partinfo-00000002-QINU
Nagahama_2015
UNIQ5a124a9e99451196-partinfo-00000003-QINU
•••
iGEM_Nagahama_2015 |
The activator of AcrAB-TolC multidrug efflux pump exports some terpenes
Export geraniol
ResistanceNagahama_2016UNIQ5a124a9e99451196-partinfo-00000005-QINU
UNIQ5a124a9e99451196-partinfo-00000007-QINU |