Motivation

An important aspect of Synthetic Biology is to prevent the uncontrolled interaction between the genetically modified organisms and the environment and mankind. For this approach there are a lot of ideas in discussion to implement different Biosafety-Systems (Wright et al., 2013) 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 (Myhr et al., 1999).
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.
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...

Antibiotic-free Selection - Introduction

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 Escherichia coli and Gram-positive Bacteria for example Bacillus subtillis. 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.
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 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).

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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.

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.
In E. coli 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. E. coli posses two alanine racemases. One, encoded by alr is constitutively expressed and therefore normally responsible for the accumulation of D-alanine, while the other one encoded by dadX is under control of the dad-operon and usually used in catabolism (Walsh, 1989).


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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) 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 BBa_K1465401. This approach can be used for an antibiotic-free selection system and even 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.
• 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.