Team:Bielefeld-CeBiTec/Results/Biosafety/Outlook

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     <h6>Remaining Challenges</h6>
     <h6>Remaining Challenges</h6>
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The <i>E. coli</i> strains KRX <i>&Delta;alr</i> <i>&Delta;dadX</i> and DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</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>
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So in conclusion it could be demonstrated, that the antibiotic-free selection system by via the D-alanine auxotrophic strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> is not only possible, but even more efficent according to the transformation efficiency with the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a>.  
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Therefore the Revertants were analyzed by streking out an overnight culture of the strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> and DH5&alpha; <i>&Delta;alr</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 3,4 10<sup>-7</sup> (overnight culture) and 3,11 10<sup>-7</sup> &plusmn; 2,29 10<sup>-7</sup> (Transformation) was estimated. Beside there was no significante difference between the revertion ratio of the strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> (3,27 10<sup>-7</sup> &plusmn; 2,27 10<sup>-7</sup>) and DH5&alpha; <i>&Delta;alr</i> <i>kan:dadX</i> (2,95 10<sup>-7</sup> &plusmn; 2,65 10<sup>-7</sup>), 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>
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A possible explantion might be a point mutation in the coding sequence of the methionine repressor <i>metJ</i>, resulting in a similar mutation rate of 7 x 10<sup>-7</sup> (<a href="#Kang2011">Kang&nbsp;<i>et&nbsp;al.</i>,&nbsp;2011</a>). Under normal circumstances the MetJ represses all essential genes for the biosynthesis of L-methionin like <i>metA, metB, metC, MetF, metE</i> and <i>metK</i> as well as the genes of the <i>metD</i> operon by using <i>S</i>-adenosylmethionine (SAM) as cofactor, see Figure x below. <br>
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Bild
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Figure X:
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It could be demonstrated that in the presence of L-methionine all genes affected are surpressed and no revertants are observable, and that the reversion could not be quantified in its absence, suggesting that there is an other methionin-repressible enzyme able to accumulate D-alanine in <i>E. coli</i>. The revertants formed in the absence of L-methionin showed a higher expression of the Cystathionin &beta;-lyase and point mutations in the MetJ repressor like R42C. It could be shwon that a strict D-alanine auxotrophy can be restore by a plasmidar expression of the natural <i>metJ</i> repressor or the additional deletion of <i>metC</i> (<a href="#Kang2011">Kang&nbsp;<i>et&nbsp;al.</i>,&nbsp;2011</a>).<br>
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Up to now the antibiotic-free selection could be demonstrated only for normal plasmids like <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> as Backbone (3163 bp) and <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> (RFP, 1069 bp), <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_I13522</a> (GFP, 937 bp) respectivley as insert, resulting in a plasmid-size of 4232 bp and 4100 bp.
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Diskussion der ratio im Vergleich zur Trafoeff und der Plasmidgröße nach GFP
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Empfehlung Methionin oder metC
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Revision as of 18:24, 16 October 2014


Biosafety

Remaining Challenges

So in conclusion it could be demonstrated, that the antibiotic-free selection system by via the D-alanine auxotrophic strain DH5α Δalr ΔdadX is not only possible, but even more efficent according to the transformation efficiency with the plasmid BBa_K1465401.

References

  • 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.

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