Difference between revisions of "Part:BBa K1653006"
Yoshiharu994 (Talk | contribs) |
(→Resistance) |
||
(16 intermediate revisions by 2 users not shown) | |||
Line 2: | Line 2: | ||
<partinfo>BBa_K1653006 short</partinfo> | <partinfo>BBa_K1653006 short</partinfo> | ||
<br> | <br> | ||
− | The activator of AcrAB-TolC multidrug efflux pump exports some | + | '''The activator of AcrAB-TolC multidrug efflux pump exports some terpenes''' |
<br>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. | <br>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 exsisting part's information of ''marA'', it gives ''E. coli'' resistance against kanamycin only. | ||
− | <br>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 | + | <br>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 toxic organic substances that are produced using bacteria. |
− | [ | + | <br> |
+ | [http://2015.igem.org/Team:Nagahama/Medal_Parts#Gold Here] is our result page. | ||
+ | <br> | ||
+ | <br> | ||
<br> | <br> | ||
Line 21: | Line 24: | ||
<partinfo>BBa_K1653006 parameters</partinfo> | <partinfo>BBa_K1653006 parameters</partinfo> | ||
<!-- --> | <!-- --> | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | 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]''' | ||
+ | |||
+ | ====Export geraniol==== | ||
+ | <br> | ||
+ | [[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> | ||
+ | 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> | ||
+ | [[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. | ||
+ | |||
+ | ]] | ||
+ | |||
+ | [[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). | ||
+ | <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). | ||
+ | ]] | ||
+ | |||
+ | |||
+ | |||
+ | |||
+ | <br> |
Latest revision as of 14:45, 17 September 2015
MarA
The activator of AcrAB-TolC multidrug efflux pump exports some terpenes
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 toxic organic substances that are produced using bacteria.
[http://2015.igem.org/Team:Nagahama/Medal_Parts#Gold Here] is our result page.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 148
- 1000COMPATIBLE WITH RFC[1000]
The following graphs show that overexpressing marA gives resistance against geraniol and decrease intracelluler geraniol concentration.
marA device BBa_K16530020
Export geraniol
Resistance