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Biosafety - Antibiotic-free Selection

Motivation

Transformation efficiency

Transformation process

Stability

Challenges

Outlook

Motivation

An important aspect of synthetic biology is to prevent the uncontrolled interaction between the genetically modified organisms, the environment and the mankind. To prevent this interaction there are many ideas in discussion to implement different biosafety systems (Wright et al., 2013) in synthetic biology. On the other hand there are several studies dealing with the interaction of genetically modified bacteria and wild types, demonstrating in most cases that genetically modified bacteria do not influence the environment. Due to the fact that genetically modified bacteria are adapted to the excellent artificial conditions of the laboratory, the wild type bacteria will usually outlast these modified strains in nature due to their better adaptation to the natural environment. Nevertheless there is always a remaining risk and no guarantee that there is really no interaction and that the release of genetically modified bacteria do not affect the equilibrium of the environment (Myhr et al., 1999) (Snow et al., 2005).
As discussed in the project description here, the most problematic factor in case of release is the transfer of synthetic or genetically modified DNA, particularly the transfer of antibiotic-resistances, which would be an active intervention in the environment.
Therefore, we want to establish an antibiotic-free selection system, which provides the possibility of using genetically modified organisms with a reduced remaining risk to the environment. Such an antibiotic-free selection system can be used for molecular cloning as well as to guarantee long-term plasmid stability. Finally, our studies revealed a further advantage of such a system. It seems to be an even more efficient selection in comparison to antibiotic based selection systems.

Antibiotic-free Selection - Introduction

The cell wall is essential for every living bacterium as it ensures stability and structure, protection against osmotic pressure and regulation of molecule transport. The composition of the cell wall differs among differnt bacteria species, a feature which is commonly used to classify taxonomy. The most common classification is based on the Gram-staining into Gram-negative, for example Escherichia coli and Gram-positive bacteria like Bacillus subtillis. Gram-negative bacteria are characterized by an inner plasma membrane, a thin peptidoglycan layer, periplasmatic spaces and the outer membrane (Figure 1). In contrast Gram-positive bacteria generally lack the outer membrane but consist of a thicker peptidoglycan layer.
Hence, the peptidoglycan layer is an interesting target to control bacterial cell dvision. Peptidoglycan itself is a polymer consisting of a linear chain of polysaccharides and short peptides. The polysaccharides component is composed of alternating residues of beta-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid. They are cross-linked in E. coli by a tetra-peptide of L-alanine, D-glutamic acid, meso-diaminopimelic acid and finally D-alanine. The cross-linkage is thereby realized by a transpeptide-linkage of meso-diaminopimelic acid and D-alanine (Cava et al., 2011).

Figure 1: Structure of the bacterial cell wall of a Gram-negative bacteria. The peptidoglycane layer consist of altering N-acetylglucosamine and N-acetylmuramic acid, which are crosslinked by a tetra-peptide. The crosslinkage is realized by D-alanine. Therefore, D-Alanin is an interesting target to control bacterial cell division.

Bacteria with a missing cross-linkage are not able to perform cell division. Cells would lysate instead because of the broken peptidoglycane layer. One possibility to prevent the crosslinkage is the use of a ß-lactam antibiotic, like penicilin. Penicillin inhibits the enzyme D-transpeptidase which is responsible for the cross linkage. In contrast to that another possibility is the prevention of the D-alanine-synthesis in the cell itself. Bacteria without D-alanine supplementation will lyse during cell division.
The synthesis of D-alanin in E. coli can be catalyzed by an alanine racemase (EC 5.1.1.1). This enzyme enables the reversible reaction from L-alanine into the enantiomer D-alanine. This reaction requires the cofactor pyridoxal-5'-phosphate (PLP) as shown in Figure 2. E. coli posseses two alanine racemases. One is encoded by alr and constitutively expressed and therefore responsible for the accumulation of D-alanine. The other one is encoded by dadX, under the control of the dad-operon and usually used in catabolism (Walsh, 1989).

Figure 2: The alanine racemase Alr from E. coli catalyses the reversible reaction from L-alanine to D-alanine, whereby Pyridoxal-5'-phosphate (PLP) is an essential cofactor for this reaction.

The deletion of the constitutive alanine racemase (alr) and the catabolic alanine racemase (dadX) in the genome will lead to a strict dependence on D-alanine. A bacterium with such a double deletion is only able to grow on media with D-alanine supplementation or with a complementation of the alanine racemase on a separate plasmid like BBa_K1465401. This approach can be used for an antibiotic-free selection system and even for molecular cloning without antibiotics.

References

Cava F, Lam H, de Pedro MA, Waldor MK (2011) Emerging knowledge of regulatory roles of d-amino acids in bacteria. Cell and Molecular Life Sciences, vol. 68, pp. 817 - 831.

Snow A., Andow D., Gepts P., Hallerman E., Power A., Tiedje J. and Wolfenbarger L. (2005) Genetically Engineered Organisms And The Environment: Current Status And Recommendations. Ecological Applications, vol. 15, pp. 377 - 404.
Myhr, Anne and Traavik, Terje (1999) The Precautionary Principle Applied to Deliberate Release of Genetically Modified Organisms (GMOs). Microbial Ecology in Health and Disease, vol. 11, pp. 65 - 74.
Walsh, Christopher (1989) Enzymes in the D-alanine branch of bacterial cell wall peptidoglycan assembly. Journal of biological chemistry, vol. 264, pp. 2393 - 2396.
Wright O, Stan GB, Ellis T. (2013) Building-in biosafety for synthetic biology. Microbiology, vol. 159, pp. 1221 - 1235.

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-<h1> Biosafety </h1>
+<h1>Biosafety - Antibiotic-free Selection</h1>
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 {{Template:Team:Bielefeld-CeBiTec/results_biosafety.tmpl}}
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 <div class="element" style="margin:10px; padding:10px">
 <div id="text">
-An important aspect of Synthetic Biology is to prevent the uncontrolled interaction between the genetically modified organismen and the environment and mankind. For this approach there are a lot of <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Project/Biosafety">ideas</a> in discussion to implement different Biosafety-Systems (<a href="#Wright2013">Wright <i>et al.</i>, 2013</a>) in synthetic Biology to prevent this interaction on one hand. On the other hand there are several studies dealing with the interaction of genetically modified bacteria and natural wild types, demonstrating in most cases that genetically modified bacteria does not influence the environment. Because the genetically modified bacteria are adapted to the excellent conditions of the laboratory, the natural bacteria will usually outlast these modified strains in nature due to their better adaptation to their environment. But there is always a risk remaining and no guarantee that there is really no interaction and that their release does not affect the equilibrium of the environment (<a href="#Myhr1999">Myhr&nbsp;<i>et&nbsp;al.</i>, 1999</a>).<br>
-So the most problematic factor in this case is the transfer of synthetic or genetically modified DNA, particularly the transfer of antibiotic-resistance, because a genetic exchange of the antibiotic-resistance is an active intervention in the environment.<br>
-Therefore we want to establish an antibiotic-free selection system, which opens the possibility of using genetically organism with a reduced remaining risk to the environment, due to the antibiotic-free selection. This antibiotic-free selection system can be used for molecular cloning as well as long-term plasmid stability and on top it turned out, that it is even more efficient than the selection using the antibiotic chloramphenicol...<br>
-<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
-  <div id="text">
-     <h6>Antibiotic-free Selection - Introduction</h6>
-       <p>
-The cell wall is essential for every living bacteria as it confers stability and structure, protects against osmotic pressure and regulates the transport of molecules.
-The composition of the cell wall differs between bacteria, a feature commonly used in taxonomy. The most common division is based on the Gram-staining into Gram-negative, for example <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli"><i>Escherichia coli</i></a> and Gram-positive Bacteria for example <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#B.subtilis"><i>Bacillus subtillis</i></a>. Gram-negative bacteria are characterized by an inner plasma membrane, a thin peptidoglycan layer, periplasmatic spaces and the outer membrane (see Figure 1). In contrast Gram-positive bacteria generally lack the outer membrane but have a thicker peptidoglycan layer.<br>
-Hence, the peptidoglycan layer is an interesting approach to control bacterial cell dvision. Peptidoglycan itself is a polymer consisting of a linear chain of polysaccharides and short peptides. The polysaccharides component are of alternating residues of beta-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid and they are cross-linked in <i>E. coli</i> by a tetra-peptide of L-alanine, D-glutamic acid, <i>meso</i>-diaminopimelic acid and finally D-alanine. The cross-linkage is thereby realized by a transpeptide-linkage of <i>meso</i>-diaminopimelic acid and D-alanine (<a href="#Cava2011">Cava <i>et al.</i>, 2011</a>).<br>
 <br>
+<h6>Motivation</h6>
-Bild
+An important aspect of synthetic biology is to prevent the uncontrolled interaction between the genetically modified organisms, the environment and the mankind. To prevent this interaction there are many <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Project/Biosafety">ideas</a> in discussion to implement different biosafety systems (<a href="#Wright2013">Wright <i>et al.</i>, 2013</a>) in synthetic biology. On the other hand there are several studies dealing with the interaction of genetically modified bacteria and wild types, demonstrating in most cases that genetically modified bacteria do not influence the environment. Due to the fact that genetically modified bacteria are adapted to the excellent artificial conditions of the laboratory, the wild type bacteria will usually outlast these modified strains in nature due to their better adaptation to the natural environment. Nevertheless there is always a remaining risk and no guarantee that there is really no interaction and that the release of genetically modified bacteria do not affect the equilibrium of the environment (<a href="#Myhr1999">Myhr&nbsp;<i>et&nbsp;al.</i>, 1999</a>) (<a href="#Snow2005">Snow&nbsp;<i>et&nbsp;al.</i>,&nbsp;2005</a>).
 <br>
+As discussed in the project description <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Project/Biosafety">here,</a> the most problematic factor in case of release is the transfer of synthetic or genetically modified DNA, particularly the transfer of antibiotic-resistances, which would be an active intervention in the environment.
-Figure 1: Structure of the bacterial cell wall of a Gram-negative bacteria. The peptidoglycane layer consist of altering N-acetylglucosamine and N-acetylmuramic acid, which are crosslinked by a tetra-peptide. The crosslinkage is realized with D-alanine and therefore an interesting approach to control bacterial cell division.<br>
 <br>
+Therefore, we want to establish an antibiotic-free selection system, which provides the possibility of using genetically modified organisms with a reduced remaining risk to the environment. Such an antibiotic-free selection system can be used for molecular cloning as well as to guarantee long-term plasmid stability. Finally, our studies revealed a further advantage of such a system. It seems to be an even more efficient selection in comparison to antibiotic based selection systems.
-Bacteria with a missing cross-linkage are not able to divide, because they will lysate instead because of the broken peptidoglycane layer. So one possiblity to prevent the crosslinkage is the use of a ß-lactam Antibiotic, like Penicilines, which inhibits the enzyme DD-transpeptidase responsible for the cross-linkage. But another possibility is the prevention of the D-alanine-synthesis in the cell itself, because bacteria without the supply of D-alanine will lyse during cell division.<br>
-In <i>E. coli</i> the accumulation of D-alanine can be catalyzed by an alanine racemase (EC 5.1.1.1). This enzyme enables the reversible reaction from L-alanine into the enantiomer D-alanine. For this reaction the cofactor pyridoxal-5'-phosphate (PLP) is also needed as shown in Figure 2. <i>E. coli</i> posses two alanine racemases. One, encoded by <i>alr</i> is constitutively expressed and therefore normally responsible for the accumulation of D-alanine, while the other one encoded by <i>dadX</i> is under control of the <i>dad</i>-operon and usually used in catabolism (<a href="#Walsh1989">Walsh, 1989</a>).<br>
 <br>
-Bild
 <br>
-Figure 2: The alanine racemase Alr from <i>E. coli</i> catalyses the reversible reaction from L-alanine to D-alanine, whereby Pyridoxal-5'-phosphate (PLP) is an essential cofactor for this reaction.<br>
+<div class="element" style="margin:10px; padding:10px">
+<div id="text">
+<h6>Antibiotic-free Selection - Introduction</h6>
+<p>
+The cell wall is essential for every living bacterium as it ensures stability and structure, protection against osmotic pressure and regulation of molecule transport.
+The composition of the cell wall differs among differnt bacteria species, a feature which is commonly used to classify taxonomy. The most common classification is based on the Gram-staining into Gram-negative, for example <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#E.coli"><i>Escherichia&nbsp;coli</i></a> and Gram-positive bacteria like <a href="https://2014.igem.org/Team:Bielefeld-CeBiTec/Notebook/Organisms#B.subtilis"><i>Bacillus&nbsp;subtillis</i></a>. Gram-negative bacteria are characterized by an inner plasma membrane, a thin peptidoglycan layer, periplasmatic spaces and the outer membrane (Figure 1). In contrast Gram-positive bacteria generally lack the outer membrane but consist of a thicker peptidoglycan layer.
 <br>
-The deletion of the constitutive alanine racemase (<i>alr</i>) and the catabolic alanine racemase (<i>dadX</i>) will lead to a strict dependence of D-alanine, so that the bacteria with these double deletion are only able to grow on media with D-alanine supplementation or by complementation of the alanine racemase on a separate plasmid like <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a>. This approach can be used for an antibiotic-free selection system and even molecular cloning without antibiotics.<br>
+Hence, the peptidoglycan layer is an interesting target to control bacterial cell dvision. Peptidoglycan itself is a polymer consisting of a linear chain of polysaccharides and short peptides. The polysaccharides component is composed of alternating residues of beta-(1,4) linked N-acetylglucosamine and N-acetylmuramic acid. They are cross-linked in <i>E.&nbsp;coli</i> by a tetra-peptide of L-alanine, D-glutamic acid, <i>meso</i>-diaminopimelic acid and finally D-alanine. The cross-linkage is thereby realized by a transpeptide-linkage of <i>meso</i>-diaminopimelic acid and D-alanine (<a href="#Cava2011">Cava <i>et al.</i>, 2011</a>).<br>
-       </p>
-  </div>
-</div>
-<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
-  <div id="text">
-     <h6>Transformation efficiency</h6>
-       <p>
-The double deletion of the DH5&alpha; and KRX strain, reuslting in the phenotypes KRX <i>&Delta;alr</i> <i>&Delta;dadX</i> and DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> respectivly. 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>
-The plasmid <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a> used for the complementation carries the konstitutive 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>
-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 supplemntation and in SOC-Media with supplemtation of 3 mM D-alanine to maintian the cell viability of the transformans, 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 antibiotica-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>
-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>
 <center>
 <div class="element" style="width:600px">
-     <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>
+     <a href="https://static.igem.org/mediawiki/2014/0/0f/Bielefeld-CeBiTec_2014-10-17_Peptidoglycan.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/0/0f/Bielefeld-CeBiTec_2014-10-17_Peptidoglycan.png" width="600px" align="center"></a><br>
-<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>
+<font size="2" style=""><b>Figure 1:</b> Structure of the bacterial cell wall of a Gram-negative bacteria. The peptidoglycane layer consist of altering N-acetylglucosamine and N-acetylmuramic acid, which are crosslinked by a tetra-peptide. The crosslinkage is realized by D-alanine. Therefore, D-Alanin is an interesting target to control bacterial cell division.</font>
 </div>
 </center>
+<br>
-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.
+Bacteria with a missing cross-linkage are not able to perform cell division. Cells would lysate instead because of the broken peptidoglycane layer. One possibility to prevent the crosslinkage is the use of a ß-lactam antibiotic, like penicilin. Penicillin inhibits the enzyme D-transpeptidase which is responsible for the cross linkage. In contrast to that another possibility is the prevention of the D-alanine-synthesis in the cell itself. Bacteria without D-alanine supplementation will lyse during cell division.
-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 contaminations 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>
+<br>
-The most interesting aspect turns out by the comparision between the chloramphenicol selection and the antibiotica-free selection (AB-free), because it can be clearly seen, that the amount of positive red colonies is 2,88 +-0,45 (3 mM D-alanine supplemtation) and 5,91 +- 1,53 respectivly (without D-alanine) fold higher compared to the selection with the antibiotic chloramphenicol. This might be due to the active inhibtion 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.
-<center>
-<div class="element" style="width:600px">
-    <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>
-<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>
-</div>
-</center>
-<center>
+The synthesis of D-alanin in <i>E.&nbsp;coli</i> can be catalyzed by an alanine racemase (EC 5.1.1.1). This enzyme enables the reversible reaction from L-alanine into the enantiomer D-alanine. This reaction requires the cofactor pyridoxal-5'-phosphate (PLP) as shown in Figure 2. <i>E.&nbsp;coli</i> posseses two alanine racemases. One is encoded by <i>alr</i> and constitutively expressed and therefore responsible for the accumulation of D-alanine. The other one is encoded by <i>dadX</i>, under the control of the <i>dad</i>-operon and usually used in catabolism (<a href="#Walsh1989">Walsh, 1989</a>).<br>
-<div class="element" style="width:600px">
-    <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>
-<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>
-</div>
-</center>
-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 spontane chloramphenicol-resistant, contaminations 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>
 <center>
 <div class="element" style="width:600px">
-     <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/2014/9/95/Bielefeld-CeBiTec_2014-10-15_Reaktion_alr.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/9/95/Bielefeld-CeBiTec_2014-10-15_Reaktion_alr.png" width="600px" align="center"></a><br>
-<font size="2" style=""><b>Figure 6:</b> Portion of false-positive colonies of classical antibiotic-selection using chlormaphenicol (red&nbsp;bars) and the antibiotical-free approach (orange bars). The false-positve colonies have been identified by their white phenotype. For the antiobiotic 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>
+<font size="2" style=""><b>Figure 2:</b> The alanine racemase Alr from <i>E.&nbsp;coli</i> catalyses the reversible reaction from L-alanine to D-alanine, whereby Pyridoxal-5'-phosphate (PLP) is an essential cofactor for this reaction.</font>
 </div>
 </center>
-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 valide transformations efficiency by using equation (1):<br>
+<br>
-Transformation effiency [cfu/ng] = colony forming units / (Volume plated out [µl]/total volume [µl])*Amount of Plasmid-DNA [ng]<br>
+The deletion of the constitutive alanine racemase (<i>alr</i>) and the catabolic alanine racemase (<i>dadX</i>) in the genome will lead to a strict dependence on D-alanine. A bacterium with such a double deletion is only able to grow on media with D-alanine supplementation or with a complementation of the alanine racemase on a separate plasmid like <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a>. This approach can be used for an antibiotic-free selection system and even for molecular cloning without antibiotics.<br>
-The estimated transformation efficiency for the transforamtion 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 shwon 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 supplemtation of D-alanine due to the lytic effect when D-alanine is lacking and the crosslinkage of the peptidoglycane can not be performed by the cell. But even thought it is still possible.
-<center>
-<div class="element" style="width:600px">
-    <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>
-<font size="2" style=""><b>Figure 7:</b> Comparision of the Transformation efficiency for the transforamtion of 0,52 ng <a href="http://parts.igem.org/Part:BBa_K1465401">BBa_K1465401</a>. The incubation was perfomed 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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-    <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>
-<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 perfomed 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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-  <div id="text">
-     <h6>Transformation process</h6>
-       <p>
-For the further investigation of the transformation process and to prove if the higher transformation efficiency is coressponding to the temporary storage of D-alanine and due to the lacking inhibition by an appropiate antibiotic the bacterial growth of the <i>E.&nbsp;coli</i> strain DH5&alpha; <i>&Delta;alr</i> <i>&Delta;dadX</i> druing incubation in the SOC-Media was analyzed. Therefore small volumes of 40 µl were continous plated out in a time intervall of 15 min. The samples were streaked out in parallel on normal LB-Agar for an antibiotic-free selection and on LB containing 30 mg/L Chlormaphenicol for the classical selection via antibiotica.<br>
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-<div class="element" style="width:600px">
-    <a href="https://static.igem.org/mediawiki/2014/c/c6/Transformation_time.png" target="_blank"><img src="https://static.igem.org/mediawiki/2014/c/c6/Transformation_time.png" width="600px" align="center"></a>
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-</center>
-to further investigate ein zeitlicher verlauf aufgenommen
-quotient of false-positive laways by about 2% as described above
-Räuber Hotzenplotz
-       </p>
-  </div>
-</div>
 <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
    <div id="text">
-      <h6>Molecular cloning without antibiotics</h6>
+      <h6>References</h6>
         <p>
-GFP
+<ul>
-       </p>
-  </div>
-</div>
+<li id="Cava2011">
 <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
    <div id="text">
-     <h6>Long-term stability of the antiobiotic-free selection</h6>
+       Cava F, Lam H, de Pedro MA, Waldor MK (2011) Emerging knowledge of regulatory roles of d-amino acids in bacteria. <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037491/pdf/18_2010_Article_571.pdf" target="_blank"><i>Cell and Molecular Life Sciences</i></a>, vol. 68, pp. 817 - 831.
-       <p>
-Graphik
-       </p>
    </div>
 </div>
+</li>
-<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
-  <div id="text">
-     <h6>Remaining Challenges</h6>
-       <p>
-MetC
-Reversion
-       </p>
-  </div>
-</div>
-<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
-  <div id="text">
-     <h6>Summary</h6>
-       <p>
-       </p>
-  </div>
-</div>
-<div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
-  <div id="text">
-     <h6>References</h6>
-       <p>
 <ul>
-<li id="Cava2011">
+<li id="Snow2005">
 <div class="element" style="margin:10px 10px 10px 10px; padding:10px 10px 10px 10px">
    <div id="text">
-        Cava F, Lam H, de Pedro MA, Waldor MK (2011) Emerging knowledge of regulatory roles of d-amino acids in bacteria. <a href="http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3037491/pdf/18_2010_Article_571.pdf" target="_blank"><i>Cell and Molecular Life Sciences</i></a>, vol. 68, pp. 817 - 831.
+        Snow A., Andow D., Gepts P., Hallerman E., Power A., Tiedje J. and Wolfenbarger L. (2005) Genetically Engineered Organisms And The Environment: Current Status And Recommendations. <a href="http://dx.doi.org/10.1890/04-0539" target="_blank"><i>Ecological Applications</i></a>, vol. 15, pp. 377 - 404.
    </div>
 </div>