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Team:Bielefeld-CeBiTec/Results/Biosafety/Outlook
From 2014.igem.org
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The <i>E. coli</i> strains KRX <i>Δalr</i> <i>ΔdadX</i> and DH5α <i>Δalr</i> <i>ΔdadX</i> respectivly showed a strict dependance of D-alanine but as mentioned above the ratio of false-positive was slightly higher compared to the selection on the antibiotic selection using Chlormaphenicol and even on the negative plate some colony forming untis were obervable, while there were no on the LB plate containing 30 mg/L Chloramphenicol. This effect migth due to some revertants of the D-alanine auxotropy and the corresponding selection pressure.<br> | The <i>E. coli</i> strains KRX <i>Δalr</i> <i>ΔdadX</i> and DH5α <i>Δalr</i> <i>ΔdadX</i> respectivly showed a strict dependance of D-alanine but as mentioned above the ratio of false-positive was slightly higher compared to the selection on the antibiotic selection using Chlormaphenicol and even on the negative plate some colony forming untis were obervable, while there were no on the LB plate containing 30 mg/L Chloramphenicol. This effect migth due to some revertants of the D-alanine auxotropy and the corresponding selection pressure.<br> | ||
| - | Therefore the Revertants were analyzed by streking out an overnight culture of the strain DH5α <i>Δalr</i> <i>ΔdadX</i> and DH5α <i>Δalr</i> <i>kan:dadX</i> on normal LB and several dilution on LB medium containing 5 mM D-alanine. The same procedure was performed with the transformation approach. In both cases the nearly the same revertants rate of xx (overnight culture) and yy (Transformation) was estimated | + | Therefore the Revertants were analyzed by streking out an overnight culture of the strain DH5α <i>Δalr</i> <i>ΔdadX</i> and DH5α <i>Δalr</i> <i>kan:dadX</i> on normal LB and several dilution on LB medium containing 5 mM D-alanine. The same procedure was performed with the transformation approach. In both cases the nearly the same revertants rate of xx (overnight culture) and yy (Transformation) was estimated. Beside there was no significante difference between the revertion ratio of the strain DH5α <i>Δalr</i> <i>ΔdadX</i> and DH5α <i>Δalr</i> <i>kan:dadX</i>, so that an effect by some contamination could be excluded and so the additional colonies probably some revertants which are able to accumulate D-alanine in some way.<br> |
| - | + | An possible explanation might be MetC (<a href="#Kang2011">Kang <i>et al.</i>, 2011</a>) | |
| - | + | ||
| - | MetC | + | |
| - | + | ||
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Revision as of 13:25, 16 October 2014
Biosafety
Remaining Challenges
The E. coli strains KRX Δalr ΔdadX and DH5α Δalr ΔdadX respectivly showed a strict dependance of D-alanine but as mentioned above the ratio of false-positive was slightly higher compared to the selection on the antibiotic selection using Chlormaphenicol and even on the negative plate some colony forming untis were obervable, while there were no on the LB plate containing 30 mg/L Chloramphenicol. This effect migth due to some revertants of the D-alanine auxotropy and the corresponding selection pressure.
Therefore the Revertants were analyzed by streking out an overnight culture of the strain DH5α Δalr ΔdadX and DH5α Δalr kan:dadX on normal LB and several dilution on LB medium containing 5 mM D-alanine. The same procedure was performed with the transformation approach. In both cases the nearly the same revertants rate of xx (overnight culture) and yy (Transformation) was estimated. Beside there was no significante difference between the revertion ratio of the strain DH5α Δalr ΔdadX and DH5α Δalr kan:dadX, so that an effect by some contamination could be excluded and so the additional colonies probably some revertants which are able to accumulate D-alanine in some way.
An possible explanation might be MetC (Kang et al., 2011)
References
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Kang L, Shaw AC, Xu D, Xia W, Zhang J, Deng J, Wöldike HF, Liu Y, Su J. (2011) Upregulation of MetC is essential for D-alanine-independent growth of an alr/dadX-deficient Escherichia coli strain. Journal of bacteriology, vol. 193, pp. 1098 - 1106.
