Difference between revisions of "Part:BBa K1653006"
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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 toxic organic substances that are produced using bacteria.  
 
<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.
 
[http://2015.igem.org/Team:Nagahama/Medal_Parts#Gold Here] is our result page.
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<br>
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<br>
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<br>
  
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<!-- Add more about the biology of this part here
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===Usage and Biology===
  
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<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K1653006 SequenceAndFeatures</partinfo>
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<!-- Uncomment this to enable Functional Parameter display
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===Functional Parameters===
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<partinfo>BBa_K1653006 parameters</partinfo>
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The following graphs show that overexpressing marA gives resistance against geraniol and decrease intracelluler geraniol concentration.
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<br>'''[https://parts.igem.org/Part:BBa_K1653020 ''marA'' device BBa_K16530020]'''
  
 
====Export geraniol====
 
====Export geraniol====
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<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'').
 
[[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.]]
  
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====Resistance====
 
<br>
 
<br>
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The following graphs show that overexpressing marA gives resistance against geraniol and decrease intracelluler geraniol concentration.
+
[[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.  
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<br>'''[https://parts.igem.org/Part:BBa_K1653020 ''marA'' device BBa_K16530020]'''
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<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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====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.
 
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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 geraniol solutions, 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 geraniol 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.  
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[[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).  
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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>
 +
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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<!-- Add more about the biology of this part here
 
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===Usage and Biology===
 
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<span class='h3bb'>Sequence and Features</span>
 
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<partinfo>BBa_K1653006 SequenceAndFeatures</partinfo>
 
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<!-- Uncomment this to enable Functional Parameter display
 
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===Functional Parameters===
 
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<partinfo>BBa_K1653006 parameters</partinfo>
 
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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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 148
  • 1000
    COMPATIBLE 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


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.

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).