Team:TU Delft/Project/tolerance/results

From 2010.igem.org

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(Solvent tolerance)
(Survival Results & Conclusions)
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==Survival Resuls & Conclusions==
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==Survival Results & Conclusions==
===Solvent tolerance===
===Solvent tolerance===
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The solvent tolerance cluster ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406]) was expressed on in ''E. coli'' K12.  The growth of the cells was tested under different amounts of n-hexane. The results suggest that this part gives to the cells an growth advantage when there's a lot of n-hexane around. The parental strain ''E. coli'' K12 seems to grow really slow at 10% (v/v) of n-hexane/M9 mixture.
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The solvent tolerance cluster ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398406 BBa_K398406]) was expressed in ''E. coli'' K12.  The growth rate of cells was challenged by different amounts of n-hexane. The results (Fig. 1) suggest that this part indeed improves growth under high n-hexane conditions. The parental strain ''E. coli'' K12 was growing very slowly at 10% (v/v) of n-hexane/M9 mixture.
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[[Image:TU_Delft_406_K12.jpg|500px|thumb|center|Growth of ''E. coli'' K12 in M9 medium at different n-hexane concentrations. ]]
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[[Image:TU_Delft_406_K12.jpg|500px|thumb|center|Fig. 1: Growth of ''E. coli'' K12 in M9 medium at different n-hexane concentrations. ]]
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Please note the growth of ''E. coli'' 406 - shows no difficulties under these harsh conditions.
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Whereas our ''E. coli'' 406 doesn't have any problem at all.
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[[Image:TU_Delft_406_406.jpg|500px|thumb|center|Fig. 2: Growth of ''E. coli'' 406A (expressing [http://partsregistry.org/Part:BBa_K398406 BBa_K398406] in pSB1A2) in M9 medium at different n-hexane concentrations. ]]
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[[Image:TU_Delft_406_406.jpg|500px|thumb|center|Growth of ''E. coli'' 406A (expressing [http://partsregistry.org/Part:BBa_K398406 BBa_K398406] in pSB1A2) in M9 medium at different n-hexane concentrations. ]]
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The growth rates were determined from the exponential phase (using a trendline, Fig. 3).
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[[Image:TU_Delft_Solvent_tolerance.jpg|500px|thumb|center|Fig. 3: Comparison of the growth rates between ''E. coli'' K12 and ''E. coli'' 406A at different n-hexane concentrations in M9 medium. ]]
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The growth rates were determined for the exponential fase (using a trendline).
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===Salt tolerance===
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The results are visible in the following chart.
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[[Image:TU_Delft_Solvent_tolerance.jpg|500px|thumb|center|Comparison of the growth rates between ''E. coli'' K12 and ''E. coli'' 406A at different n-hexane concentrations in M9 medium. ]]
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We tested the growth of our bbc1 construct ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108]) under different [http://2010.igem.org/Team:TU_Delft#page=Project/tolerance/characterization salt concentrations].
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===Salt tolerance===
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The growth rates were determined for the exponential phase (using a trendline, Fig. 4).
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We tested the growth of our bbc1 construct ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108]) as stated in our characterization page.
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[[Image:TU_Delft_Salt_tolerance.jpg|500px|thumb|center|Fig. 4: Growth rate in dependence of salt (NaCl).]]
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The growth rates were determined for the exponential fase (using a trendline).
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Up to 0.2 M NaCl effects of salt stress are equally observed for both the negative control and for cells with our biobrick ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108]). At higher concentrations a significant improvement of growth rate in comparison to the control background ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398027 BBa_K398027]) is seen.  
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The results are visible in the following chart.
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[[Image:TU_Delft_Salt_tolerance.jpg|500px|center]]
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===Conclusions===
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While at low salt concentrations no phenotype is observed, the resistance to high salt concentrations is significantly improved (up to 35%). The observed behavior can be explained by the vast amount of effects resulting from salt stress. It is possible that our BioBrick assists to reduce one of the inhibiting effects ,and thus leading to a benefit at higher salt stress.  
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At 0.2 M some effects of the hightened increased salt stress starts to appear, at this concentration the cells with our biobrick ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398108 BBa_K398108]) shows a significant increase in growth rate in comparison to our the control background ([http://partsregistry.org/wiki/index.php?title=Part:BBa_K398027 BBa_K398027]). At higher salt concentrations the salt stress causes a multitude of effects and as our BioBrick simply counters one, our strain also shows effects of higher salt stress. In conclusion one can see that our BioBrick is indeed extends the salt tolerance of the cells functional at concentrations greater higher then 0.3 M Sodium Chloride. The general increase in tolerance varies between 10 and 35% depending on the Sodium Chloride concentration.
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It was shown, that our BioBrick indeed increases the salt tolerance for concentrations higher than 0.3 M sodium chloride. The general increase in tolerance varies between 10 and 35% depending on the sodium chloride concentration (showing a peak at 0.5 M NaCl which coincides with the concentration of NaCl in sea water).
===Future prospects===
===Future prospects===

Revision as of 20:09, 27 October 2010

CharacterizationResultsParts


Survival Results & Conclusions

Solvent tolerance

The solvent tolerance cluster (BBa_K398406) was expressed in E. coli K12. The growth rate of cells was challenged by different amounts of n-hexane. The results (Fig. 1) suggest that this part indeed improves growth under high n-hexane conditions. The parental strain E. coli K12 was growing very slowly at 10% (v/v) of n-hexane/M9 mixture.

Fig. 1: Growth of E. coli K12 in M9 medium at different n-hexane concentrations.

Please note the growth of E. coli 406 - shows no difficulties under these harsh conditions.

Fig. 2: Growth of E. coli 406A (expressing BBa_K398406 in pSB1A2) in M9 medium at different n-hexane concentrations.

The growth rates were determined from the exponential phase (using a trendline, Fig. 3).

Fig. 3: Comparison of the growth rates between E. coli K12 and E. coli 406A at different n-hexane concentrations in M9 medium.

Salt tolerance

We tested the growth of our bbc1 construct (BBa_K398108) under different salt concentrations.

The growth rates were determined for the exponential phase (using a trendline, Fig. 4).

Fig. 4: Growth rate in dependence of salt (NaCl).

Up to 0.2 M NaCl effects of salt stress are equally observed for both the negative control and for cells with our biobrick (BBa_K398108). At higher concentrations a significant improvement of growth rate in comparison to the control background (BBa_K398027) is seen.

Conclusions

While at low salt concentrations no phenotype is observed, the resistance to high salt concentrations is significantly improved (up to 35%). The observed behavior can be explained by the vast amount of effects resulting from salt stress. It is possible that our BioBrick assists to reduce one of the inhibiting effects ,and thus leading to a benefit at higher salt stress.

It was shown, that our BioBrick indeed increases the salt tolerance for concentrations higher than 0.3 M sodium chloride. The general increase in tolerance varies between 10 and 35% depending on the sodium chloride concentration (showing a peak at 0.5 M NaCl which coincides with the concentration of NaCl in sea water).

Future prospects

We hope that the salt tolerance can be increased further by adding systems (such as ion pumps) to ensure the intracellular threshold and minimize the effects of salt stress even further. As the host organism would then be able to maintain growth at lethal concentrations of both salt and alkanes. This would allow future generations of igemmers to create cultures in a variety of different media, opening a new field of possibilities.

CharacterizationResultsParts