Team:Bielefeld-CeBiTec/Results/Biosafety/TransformationEfficiency

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<h1> Biosafety </h1>
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<h1>Biosafety - Antibiotic-free Selection</h1>
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              <p class="buttoncenter"><font color="#FFFFFF">Motivation</font></p>
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               <p class="buttoncenter"><font color="#FFFFFF">Molecular cloning</font></p>
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               <p class="buttoncenter"><font color="#FFFFFF">Long-term stability</font></p>
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               <p class="buttoncenter"><font color="#FFFFFF">Stability</font></p>
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              <p class="buttoncenter"><font color="#FFFFFF">Challenges</font></p>
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     <h6>Transformation efficiency</h6>
     <h6>Transformation efficiency</h6>
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The double deletion of the DH5&alpha; and KRX strain, resulting in the phenotypes KRX <i>&Delta;alr</i> <i>&Delta;dadX</i> and DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> respectively. This leads to a strict D-alanine auxotrophy, which can be complemented by a plasmid carrying one of the alanine racemases or D-alanine supplementation in the media.<br>
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The double deletion of the constitutive alanine racemase (<i>alr</i>) and the catabolic alanine racemase (<i>dadX</i>) in the DH5&alpha; and KRX strain, resulting in the phenotypes KRX <i>&Delta;alr</i> <i>&Delta;dadX</i> and DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> respectively. This leads to a strict D-alanine auxotrophy, which can be complemented by a plasmid carrying one of the alanine racemases or D-alanine supplementation in the media.
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The plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> used for the complementation carries the constitutive alanine racemase <i>alr</i> and the Chloramphenicol acetyltransferase for the Chloramphenicol-resistance (Cm). Besides the plasmid contains RFP <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> as a reporter for the characterization of the successful complementation.<br>
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<br>
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For the proof of the antibiotic-free selection the plasmid was transformed in different concentrations into electrocompetent cells of DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i>. As it turned out, that the cell can store D-alanine for some time the incubation of the cells after the transformation was performed in normal <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#SOC-medium">SOC-Media</a> without any supplementation and in SOC-Media with supplementation of 3 mM D-alanine to maintain the cell viability of the transformants, before they are able to express the alanine racemase essential for the accumulation of D-alanine and bacterial growth. After the incubation the cells were streaked out either of LB plates for the antibiotic-free selection via complementation of the D-alanine auxotrophy or were streaked out of LB plates containing 30 mg/L Chlormaphenicol as control. The successful transformation could be verified by the red color of the reporter RFP, while false-positive colonies remain white.<br>
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The plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> which is used for the complementation carries the constitutive alanine racemase <i>alr</i> and the chloramphenicol acetyltransferase for the chloramphenicol-resistance (Cm). Besides the plasmid contains RFP <a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a> as a reporter for the characterization of the complementation efficiency.
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An example for the transformation of 0,33 ng of the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> into the <i>E. coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> is shown in Figure 3. In the upper row different volumes from 50 µl, 100 µl and 200 µl were plated on LB containing Chloramphenicol and in the lower row the same volumes were streaked out only on normal LB for the selection without any antibiotic. Surprisingly it becomes obvious that the antibiotic-free selection is more efficient than the selection via Chloramphenicol, because more colonies could be observed on each plate with an equal volume plated.<br>
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To investigate the antibiotic-free selection the plasmid was transformed in different concentrations into electrocompetent cells of DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i>. Due to the fact, that the cell can store D-alanine for a certain periode of time the incubation of the cells after the transformation was performed in normal <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#SOC-medium">SOC-media</a> without supplementation and in SOC-media with supplementation of 3 mM D-alanine to maintain the cell viability of the transformants before they are able to express the alanine racemase which is essential for the accumulation of D-alanine and bacterial growth. After the incubation the cells were stread out either onto LB plates for the antibiotic-free selection via complementation of the D-alanine auxotrophy or onto LB plates containing 30 mg/L chlormaphenicol as control. The successful transformation can be verified by the red color of the reporter RFP, while false-positive colonies remain white.
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An example for the transformation of 0.33 ng plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> into the <i>E. coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> is shown in Figure 3. The upper row shows different volumes from 50 µl, 100 µl and 200 µl were spread onto LB containing chloramphenicol and the lower row shows the same volumes were spread out onto normal LB for the selection without any antibiotic. It appears that the antibiotic-free selection is more efficient than the selection with chloramphenicol because more colonies were observed on each plate with equal volume plated.<br>
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     <a href="https://static.igem.org/mediawiki/2014/7/7b/Bielefeld-CeBiTec_2014-10-13_Biosafety_Selection.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/7/7b/Bielefeld-CeBiTec_2014-10-13_Biosafety_Selection.png" width="600px" align="center"></a><br>
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<font size="2" style=""><b>Figure 3:</b> Colony forming units (cfu) by the selection of Chloramphenicol (upper row) and antibiotic-free selection (lower row). 0,33 ng of the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> were transformed into the <i>E. coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i>, incubated in SOC-Media supplemented with 3 mM D-alanine and streaked out in different volumes from 50 µl (left), 100 µl (middle) and 200 µl (right).</font>
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<font size="2" style=""><b>Figure 3:</b> Colony forming units (cfu) for the selection with chloramphenicol (upper row) and antibiotic-free selection (lower row). 0.33 ng of the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> was transformed into the <i>E.&nbsp;coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i>, incubated in SOC-Media supplemented with 3 mM D-alanine and spread out in different volumes from 50 µl (left), 100 µl (middle) and 200 µl (right).</font>
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In Figure 3 and 4 the colony forming units (cfu) of the transformation of 0,52 ng plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> after one hour of incubation in either normal SOC-medium or supplemented with 3 mM D-alanine.  
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Figure 3 and 4 show the colony forming units (cfu) for the transformation of 0.52 ng plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> at one hour of incubation in either normal SOC-medium or SOC-medium supplemented with 3 mM D-alanine.  
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First of all it can be seen, that the incubation of the transformation is possible also without the supplementation of D-alanine (blue bars), but there are not as much colonies compared to the incubation with 3 mM D-alanine (red bars). Besides the graphics show, that in the absent of D-alanine there are more false-positive phenotypic white colonies (cyan bars) compared to the supplementation of D-alanine (purple bar). In some cases this leads also to huge contamination so that the amount of false-positive is even higher than the amount of correct colonies. This effect can clearly be seen by streaking out 200 µl of the transformants who were incubated in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#SOC-medium">SOC-Media</a> without D-alanine.<br>
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First of all it is possible to see, that the incubation of the transformation can be performed without the supplementation of D-alanine (blue bars), even though there are not as many colonies compared to the incubation with 3 mM D-alanine (red bars). Besides, the graphics show that in the absent of D-alanine more false-positive phenotypic white colonies occur (cyan bars) compared to the samples with supplementation of D-alanine (purple bar). In some cases the absence of D-alanin also leads to huge contamination an higher amount of false-positive colonies than correct colonies. This effect can be clearly seen by spreading 200 µl of the transformants incubated in <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Media#SOC-media">SOC-Media</a> without D-alanine.
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The most interesting aspect turns out by the comparision between the Chloramphenicol selection and the antibiotic-free selection (AB-free), because it can be clearly seen, that the amount of positive red colonies is 2,88 &plusmn;+-0,45 (3 mM D-alanine supplementation) and 5,91 +- 1,53 respectively (without D-alanine) fold higher compared to the selection with the antibiotic Chloramphenicol. This might be due to the active inhibition of Chlormaphenicol, while the lack of D-alanine can be compensated by intracellular storage of D-alanine or eventually by and temporary grow arrest, until the alanine racemase is expressed.
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The most interesting aspect turns out by the comparison between the chloramphenicol selection and the antibiotic-free selection (AB-free). It can be clearly seen that the amount of positive red colonies is 2.88 &plusmn; 0.45 (3 mM D-alanine supplementation) and 5.91 &plusmn; 1.53 (without D-alanine) fold higher compared to the selection with the antibiotic chloramphenicol. This might be due to the active inhibition of chlormaphenicol, whereas the lack of D-alanine can be compensated by intracellular storage of D-alanine or eventually by temporary grow arrest, until the alanine racemase is expressed.
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     <a href="https://static.igem.org/mediawiki/2014/d/d3/Bielefeld-CeBiTec_2014-10-14_Colonies_Cm.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/d/d3/Bielefeld-CeBiTec_2014-10-14_Colonies_Cm.png" width="600px" align="center"></a><br>
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<font size="2" style=""><b>Figure 4:</b> Colony forming units (cfu) by the selection of Chloramphenicol. 0,52 ng of the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> were transformed into the <i>E. coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> and incubated in normal SOC-Media or SOC-Media supplemented with 3 mM D-alanine. After one hour different volumes streaked out on LB containing 30 mg/L Chloramphenicol (n = 2 x 2).</font>
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<font size="2" style=""><b>Figure 4:</b> Colony forming units (cfu) for the selection with Chloramphenicol. 0.52&nbsp;ng of the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> were transformed into the <i>E.&nbsp;coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> and incubated in normal SOC-Media or SOC-Media supplemented with 3&nbsp;mM D-alanine. After one hour different volumes were spread onto LB plates containing 30&nbsp;mg/L chloramphenicol (n = 2 x 2).</font>
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     <a href="https://static.igem.org/mediawiki/2014/3/30/Bielefeld-CeBiTec_2014-10-15_Colonies_AB_free.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/3/30/Bielefeld-CeBiTec_2014-10-15_Colonies_AB_free.png" width="600px" align="center"></a><br>
     <a href="https://static.igem.org/mediawiki/2014/3/30/Bielefeld-CeBiTec_2014-10-15_Colonies_AB_free.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/3/30/Bielefeld-CeBiTec_2014-10-15_Colonies_AB_free.png" width="600px" align="center"></a><br>
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<font size="2" style=""><b>Figure 5:</b> Colony forming units (cfu) by the antibiotic-free selection of LB. 0,52 ng of the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> were transformed into the <i>E. coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> and incubated in normal SOC-Media or SOC-Media supplemented with 3 mM D-alanine. After one hour different volumes streaked out on normal LB-Media (n = 2 x 2).</font>
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<font size="2" style=""><b>Figure 5:</b> Colony forming units (cfu) for the antibiotic-free selection with LB. 0.52 ng of the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> was transformed into the <i>E.&nbsp;coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> and incubated in normal SOC-media or SOC-Media supplemented with 3&nbsp;mM D-alanine. After one hour different volumes were spread onto normal LB plates (n = 2 x 2).</font>
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Another important aspect of an antibiotic-free selection approach is the quotient of false-positive and partically the portion of revertants that could also reduce this quotient in a antibiotic-free selection attempt. Therefore the portion of false-positive identified by its white color to the positive red colored transformants are shown in Figure 6. Besides the false-positive transformants in the antibiotic-free selection, there could also be observed false-positive using Chloramphenicol selection. This false-positive could be bacteria with a spontaneous Chloramphenicol-resistant, contamination by bacteria with a Chloramphenicol-resistant or bacteria carrying a mutation within the expression of RFP (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>) leading also to a white phenotype. Within the antibiotic-free selection an average portion of 2,83 % +- 0,09 false-positive have been identified, while the antibiotic-selection via chloramphenicol shows only an average of 1,47 % +- 0,77 false-positive. The slightly higher rate of false-positive of the antibiotic-free selection might due to some <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/Biosafety#Remaining Challenges">revertants</a> which are able to accumulate D-alanine also without any alanine racemase by mutation and side reaction of another enzyme, but because of the higher transformation efficiency this effect is actually negligible.<br>
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Another important aspect of an antibiotic-free selection approach is the quotient of false-positive and particularly the portion of revertants which could also reduce this quotient in an antibiotic-free selection. Therefore, the ratio of false-positive transformants (white color) to the positive transformants (red color) are shown in Figure 6. Besides, it should be mentioned that the false-positive transformants in the antibiotic-free selection can be also observed for chloramphenicol selection. These false-positive transformants could be bacteria with a spontaneous chloramphenicol resistance, contamination by bacteria with a chloramphenicol resistance or bacteria carrying a mutation within the expression of RFP (<a href="http://parts.igem.org/Part:BBa_J04450">BBa_J04450</a>) leading also to a white phenotype. Within the antibiotic-free selection an average portion of 2.83 % &plusmn; 0.09 false-positive transformants has been identified, while the antibiotic-selection with chloramphenicol only shows an average of 1.47 % &plusmn; 0.77 false-positive transformants. The slightly higher rate of false-positive transformants of the antibiotic-free selection might due to some <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Results/Biosafety#Remaining Challenges">revertants</a> which are able to accumulate D-alanine without any alanine racemase by mutation and side reaction of another enzyme. Because of the higher transformation efficiency this effect is actually negligible.
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     <a href="https://static.igem.org/mediawiki/2014/4/4b/Bielefeld-CeBiTec_2014-10-13_Portion_of_false-negative.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/4/4b/Bielefeld-CeBiTec_2014-10-13_Portion_of_false-negative.png" width="600px" align="center"></a><br>
     <a href="https://static.igem.org/mediawiki/2014/4/4b/Bielefeld-CeBiTec_2014-10-13_Portion_of_false-negative.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/4/4b/Bielefeld-CeBiTec_2014-10-13_Portion_of_false-negative.png" width="600px" align="center"></a><br>
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<font size="2" style=""><b>Figure 6:</b> Portion of false-positive colonies of classical antibiotic-selection using Chlormaphenicol (red&nbsp;bars) and the antibiotic-free approach (orange bars). The false-positve colonies have been identified by their white phenotype. For the antibiotic selection an average of  1,47 % +- 0,77 false-positive were estimated, while the portion amount to 2,83 % +- 0,09 for the antibiotic-free selection system.</font>
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<font size="2" style=""><b>Figure 6:</b> Portion of false-positive colonies of antibiotic selection using chlormaphenicol (red&nbsp;bars) and the antibiotic-free approach (orange bars). The false positve colonies have been identified by their white phenotype. For the antibiotic selection an average of  1.47 % &plusmn; 0.77 false-positive colonies was investigated, whereas the portion amount for the antibiotic-free selection system is of to 2.83 % &plusmn; 0.09.</font>
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To obtain more reliable data, the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> were transformed in different concentrations (0,33&nbsp;ng;&nbsp;0,52&nbsp;ng and 0,65&nbsp;ng) and transformation attempt into the d-alanine auxotrophic <i>E.&nbsp;coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> and streaked out again in various volumes (50 µl; 100 µl and 200 µl) to estimate a valid transformations efficiency by using equation (1):<br>
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To obtain more reliable data, the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> was transformed in different concentrations (0.33&nbsp;ng;&nbsp;0.52&nbsp;ng and 0.65&nbsp;ng) and transformed into the D-alanine auxotrophic <i>E.&nbsp;coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> in various volumes (50 µl; 100 µl and 200 µl) to estimate a valid transformation efficiency by using equation (1):<br>
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Transformation efficiency [cfu/ng] = colony forming units / (Volume plated out [µl]/total volume [µl])*Amount of Plasmid-DNA [ng]<br>
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Transformation efficiency [cfu/ng] = colony forming units / (Spread volume [µl]/total volume [µl])*Amount of Plasmid-DNA [ng]<br>
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The estimated transformation efficiency for the transformation of 0,52 ng of <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> for the distinct volumes are shown in Figure 7, while the average for the transformation of different amounts of DNA are shown in Figure 8. And again it becomes clear, that the antibiotic-free selection is a lot more efficient then the selection by the antibiotic Chloramphenicol due to the inhibition of the antibiotic. And the other aspect obvious is that the transformation efficiency is reduced without the supplementation of D-alanine due to the lytic effect when D-alanine is lacking and the cross-linkage of the peptidoglycane can not be performed by the cell. But even thought it is still possible.
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The estimated transformation efficiency for the transformation of 0.52 ng of <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> for the distinct volumes is shown in Figure 7 and the average for the transformation of different amounts of DNA is shown in Figure 8. The result emphasizes, that the antibiotic-free selection is significantly more efficient than the selection with the antibiotic chloramphenicol due to the inhibition of <i>E.&nbsp;coli</i> by the antibiotic. Another important aspect is the reduced transformation efficiency without the supplementation of D-alanine due to the lytic effect when D-alanine is lacking and the cross-linkage of the peptidoglycane can not be performed by D-alanin lacking cells. Even though some cells show a growth.
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     <a href="https://static.igem.org/mediawiki/2014/7/71/Bielefeld_CeBiTec_2014-10-12_Transformation_Efficiency_08_AB_alr.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/7/71/Bielefeld_CeBiTec_2014-10-12_Transformation_Efficiency_08_AB_alr.png" width="600px" align="center"></a><br>
     <a href="https://static.igem.org/mediawiki/2014/7/71/Bielefeld_CeBiTec_2014-10-12_Transformation_Efficiency_08_AB_alr.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/7/71/Bielefeld_CeBiTec_2014-10-12_Transformation_Efficiency_08_AB_alr.png" width="600px" align="center"></a><br>
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<font size="2" style=""><b>Figure 7:</b> Comparision of the Transformation efficiency for the transformation of 0,52 ng <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a>. The incubation was performed either in normal SOC media or in SOC-media supplemented with 3 mM D-alanine and the colony forming units were counted on LB_Cm as well as normal LB for the antibiotic-free selection (n = 2 x 2).</font>
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<font size="2" style=""><b>Figure 7:</b> Comparision of the transformation efficiency for the transformation of 0.52 ng <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a>. The incubation was performed either in normal SOC media or in SOC media supplemented with 3&nbsp;mM D-alanine and the colony forming units were counted on LB_Cm as well as normal LB for the antibiotic-free selection (n = 2 x 2).</font>
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     <a href="https://static.igem.org/mediawiki/2014/0/09/Transformation_efficiency_AB_alr.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/0/09/Transformation_efficiency_AB_alr.png" width="600px" align="center"></a><br>
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<font size="2" style=""><b>Figure 8:</b> Comparision of the Transformation efficiency for different amount of the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a>. The incubation was performed either in normal SOC media or in SOC-media supplemented with 3 mM D-alanine and the colony forming units were counted on LB_Cm as well as normal LB for the antibiotic-free selection (n = 2 x 2).</font>
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<font size="2" style=""><b>Figure 8:</b> Comparision of the transformation efficiency for different amount of the plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a>. The incubation was performed either in normal SOC media or in SOC media supplemented with 3&nbsp;mM D-alanine and the colony forming units were counted on LB_Cm as well as normal LB for the antibiotic-free selection (n = 2 x 2).</font>
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Latest revision as of 00:06, 18 October 2014


Biosafety - Antibiotic-free Selection

Transformation efficiency

The double deletion of the constitutive alanine racemase (alr) and the catabolic alanine racemase (dadX) in the DH5α and KRX strain, resulting in the phenotypes KRX Δalr ΔdadX and DH5α Δalr ΔdadX respectively. This leads to a strict D-alanine auxotrophy, which can be complemented by a plasmid carrying one of the alanine racemases or D-alanine supplementation in the media.
The plasmid BBa_K1465401 which is used for the complementation carries the constitutive alanine racemase alr and the chloramphenicol acetyltransferase for the chloramphenicol-resistance (Cm). Besides the plasmid contains RFP BBa_J04450 as a reporter for the characterization of the complementation efficiency.
To investigate the antibiotic-free selection the plasmid was transformed in different concentrations into electrocompetent cells of DH5α Δalr ΔdadX. Due to the fact, that the cell can store D-alanine for a certain periode of time the incubation of the cells after the transformation was performed in normal SOC-media without supplementation and in SOC-media with supplementation of 3 mM D-alanine to maintain the cell viability of the transformants before they are able to express the alanine racemase which is essential for the accumulation of D-alanine and bacterial growth. After the incubation the cells were stread out either onto LB plates for the antibiotic-free selection via complementation of the D-alanine auxotrophy or onto LB plates containing 30 mg/L chlormaphenicol as control. The successful transformation can be verified by the red color of the reporter RFP, while false-positive colonies remain white.
An example for the transformation of 0.33 ng plasmid BBa_K1465401 into the E. coli strain DH5α Δalr ΔdadX is shown in Figure 3. The upper row shows different volumes from 50 µl, 100 µl and 200 µl were spread onto LB containing chloramphenicol and the lower row shows the same volumes were spread out onto normal LB for the selection without any antibiotic. It appears that the antibiotic-free selection is more efficient than the selection with chloramphenicol because more colonies were observed on each plate with equal volume plated.


Figure 3: Colony forming units (cfu) for the selection with chloramphenicol (upper row) and antibiotic-free selection (lower row). 0.33 ng of the plasmid BBa_K1465401 was transformed into the E. coli strain DH5α Δalr ΔdadX, incubated in SOC-Media supplemented with 3 mM D-alanine and spread out in different volumes from 50 µl (left), 100 µl (middle) and 200 µl (right).
Figure 3 and 4 show the colony forming units (cfu) for the transformation of 0.52 ng plasmid BBa_K1465401 at one hour of incubation in either normal SOC-medium or SOC-medium supplemented with 3 mM D-alanine. First of all it is possible to see, that the incubation of the transformation can be performed without the supplementation of D-alanine (blue bars), even though there are not as many colonies compared to the incubation with 3 mM D-alanine (red bars). Besides, the graphics show that in the absent of D-alanine more false-positive phenotypic white colonies occur (cyan bars) compared to the samples with supplementation of D-alanine (purple bar). In some cases the absence of D-alanin also leads to huge contamination an higher amount of false-positive colonies than correct colonies. This effect can be clearly seen by spreading 200 µl of the transformants incubated in SOC-Media without D-alanine.
The most interesting aspect turns out by the comparison between the chloramphenicol selection and the antibiotic-free selection (AB-free). It can be clearly seen that the amount of positive red colonies is 2.88 ± 0.45 (3 mM D-alanine supplementation) and 5.91 ± 1.53 (without D-alanine) fold higher compared to the selection with the antibiotic chloramphenicol. This might be due to the active inhibition of chlormaphenicol, whereas the lack of D-alanine can be compensated by intracellular storage of D-alanine or eventually by temporary grow arrest, until the alanine racemase is expressed.

Figure 4: Colony forming units (cfu) for the selection with Chloramphenicol. 0.52 ng of the plasmid BBa_K1465401 were transformed into the E. coli strain DH5α Δalr ΔdadX and incubated in normal SOC-Media or SOC-Media supplemented with 3 mM D-alanine. After one hour different volumes were spread onto LB plates containing 30 mg/L chloramphenicol (n = 2 x 2).


Figure 5: Colony forming units (cfu) for the antibiotic-free selection with LB. 0.52 ng of the plasmid BBa_K1465401 was transformed into the E. coli strain DH5α Δalr ΔdadX and incubated in normal SOC-media or SOC-Media supplemented with 3 mM D-alanine. After one hour different volumes were spread onto normal LB plates (n = 2 x 2).
Another important aspect of an antibiotic-free selection approach is the quotient of false-positive and particularly the portion of revertants which could also reduce this quotient in an antibiotic-free selection. Therefore, the ratio of false-positive transformants (white color) to the positive transformants (red color) are shown in Figure 6. Besides, it should be mentioned that the false-positive transformants in the antibiotic-free selection can be also observed for chloramphenicol selection. These false-positive transformants could be bacteria with a spontaneous chloramphenicol resistance, contamination by bacteria with a chloramphenicol resistance or bacteria carrying a mutation within the expression of RFP (BBa_J04450) leading also to a white phenotype. Within the antibiotic-free selection an average portion of 2.83 % ± 0.09 false-positive transformants has been identified, while the antibiotic-selection with chloramphenicol only shows an average of 1.47 % ± 0.77 false-positive transformants. The slightly higher rate of false-positive transformants of the antibiotic-free selection might due to some revertants which are able to accumulate D-alanine without any alanine racemase by mutation and side reaction of another enzyme. Because of the higher transformation efficiency this effect is actually negligible.

Figure 6: Portion of false-positive colonies of antibiotic selection using chlormaphenicol (red bars) and the antibiotic-free approach (orange bars). The false positve colonies have been identified by their white phenotype. For the antibiotic selection an average of 1.47 % ± 0.77 false-positive colonies was investigated, whereas the portion amount for the antibiotic-free selection system is of to 2.83 % ± 0.09.

To obtain more reliable data, the plasmid BBa_K1465401 was transformed in different concentrations (0.33 ng; 0.52 ng and 0.65 ng) and transformed into the D-alanine auxotrophic E. coli strain DH5α Δalr ΔdadX in various volumes (50 µl; 100 µl and 200 µl) to estimate a valid transformation efficiency by using equation (1):
Transformation efficiency [cfu/ng] = colony forming units / (Spread volume [µl]/total volume [µl])*Amount of Plasmid-DNA [ng]
The estimated transformation efficiency for the transformation of 0.52 ng of BBa_K1465401 for the distinct volumes is shown in Figure 7 and the average for the transformation of different amounts of DNA is shown in Figure 8. The result emphasizes, that the antibiotic-free selection is significantly more efficient than the selection with the antibiotic chloramphenicol due to the inhibition of E. coli by the antibiotic. Another important aspect is the reduced transformation efficiency without the supplementation of D-alanine due to the lytic effect when D-alanine is lacking and the cross-linkage of the peptidoglycane can not be performed by D-alanin lacking cells. Even though some cells show a growth.

Figure 7: Comparision of the transformation efficiency for the transformation of 0.52 ng BBa_K1465401. The incubation was performed either in normal SOC media or in SOC media supplemented with 3 mM D-alanine and the colony forming units were counted on LB_Cm as well as normal LB for the antibiotic-free selection (n = 2 x 2).

Figure 8: Comparision of the transformation efficiency for different amount of the plasmid BBa_K1465401. The incubation was performed either in normal SOC media or in SOC media supplemented with 3 mM D-alanine and the colony forming units were counted on LB_Cm as well as normal LB for the antibiotic-free selection (n = 2 x 2).