Difference between revisions of "Part:BBa K3861003"

Revision as of 17:34, 19 October 2021

ThyA CDS

The coding region of the Thymidylate synthase (ThyA, EC2.1.1.45). The enzyme catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to 2'-deoxythymidine-5'-monophosphate (dTMP). Salmonella typhimurium thyA knock out mutants cannot synthesize DNA if no thymidine monophosphate (dTMP) is available to the cells¹. Furthermore, thyA mutants were shown to not grow intercellular in macrophage-like and human epithelial cell lines in vitro². Thus, for dTMP auxotrophic bacterial strains can be created. We used this knowledge to our advantage and used thyA as selection marker. By deleting the chromosomal thyA gene and reintroducing it with one of our plasmids (BBa_K3861005), we effectively bypassed the need for an antibiotic based selection marker system.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

References

1. Neuhard, J. & Kelln, R. A. (1996). Biosynthesis and conversions ofpyrimidines. In Escherichia coli and Salmonella, pp. 580–599. American Society for Microbiology 2. Kok, M., Bühlmann, E. & Pechère, J.-C. 2001. Salmonella typhimurium thyA mutants fail to grow intracellularly in vitro and are attenuated in mice. Microbiology 147, 727–733.

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-The coding region of the Thymidylate synthase (ThyA, EC2.1.1.45). The enzyme catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to 2'-deoxythymidine-5'-monophosphate (dTMP). <i>Salmonella</i> typhimurium <i>thyA</i> knock out mutants cannot synthesize DNA if no thymidine monophosphate (dTMP) is available to the cells (Neuhard and Kelln, 1996). Furthermore, <i>thyA</i> mutants were shown to not grow intercellular in macrophage-like and  human epithelial cell lines <i>in vitro</i> (Kok et al., 2001). Thus, for dTMP auxotrophic bacterial strains can be created. We used this knowledge to our advantage and used <i>thyA</i> as selection marker. By deleting the chromosomal <i>thyA</i> gene and reintroducing it with one of our plasmids (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K3861005">pKF01</a>), we effectively bypassed the need for an antibiotic based selection marker system.
+The coding region of the Thymidylate synthase (ThyA, EC2.1.1.45). The enzyme catalyzes the reductive methylation of 2'-deoxyuridine-5'-monophosphate (dUMP) to 2'-deoxythymidine-5'-monophosphate (dTMP). <i>Salmonella</i> typhimurium <i>thyA</i> knock out mutants cannot synthesize DNA if no thymidine monophosphate (dTMP) is available to the cells<sup>1</sup>. Furthermore, <i>thyA</i> mutants were shown to not grow intercellular in macrophage-like and  human epithelial cell lines <i>in vitro</i><sup>2</sup>. Thus, for dTMP auxotrophic bacterial strains can be created. We used this knowledge to our advantage and used <i>thyA</i> as selection marker. By deleting the chromosomal <i>thyA</i> gene and reintroducing it with one of our plasmids (<a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K3861005">BBa_K3861005</a>), we effectively bypassed the need for an antibiotic based selection marker system.
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 ==='''References'''===
+.      Neuhard, J. & Kelln, R. A. (1996). Biosynthesis and conversions ofpyrimidines. In Escherichia coli and Salmonella, pp. 580–599. <i>American Society for Microbiology</i>
+.	Kok, M., Bühlmann, E. & Pechère, J.-C. 2001. Salmonella typhimurium thyA mutants fail to grow intracellularly in vitro and are attenuated in mice. <i>Microbiology 147</i>, 727–733.