Coding

Part:BBa_K1470001

Designed by: Pascal Sartor   Group: iGEM14_Freiburg   (2014-10-01)

puromycin N-acetyl-transferase

Puromycin is an antibodic derived from the bacteria Streptomyces alboniger. The substance affects the cell both in protein translation and mitochondrial protein import [1]. Due to this, it's toxic to many organisms such as bacteria, Trypanosoma and humans.
A small part of its structure resembles to aminoacetylated tRNA and is recognized by ribosomes. The nascent chain containing puromycin leads to a translational stop.
Streptomyces alboniger protects itself from puromycin with an acetyl transferase. It uses acetyl-CoA to bind the acetyl moiety to the amino group. In this state puromycin cannot be recognized by the ribosom anymore [2].
This enzyme is often used for selecting stable eucaryotic cell lines. Because of the blocked protein biosynthesis more than 99 % of uninfected cells will be dead within two days. We transduced HEK-293T cells via viral vectors including puromycin N-acetyl-transferase as a selection marker. After incubating the cells in medium with 0,5 µg /mL up to 2 µg /mL Puromycin for two days, transduced cells were vital. Hence untransduced cells, which served as a control died within two days.

References

[1] NEUBERT, D. AND LEHNINGER, A. L.: The effect of oligomycin, gramicidin, and other antibiotics on reversal of mitochondrial swelling by adenosine triphosphate. Biochim. biophys. Acta 62: 556-565, 1962.
[2] DARKEN MA.: Puromycin inhibition of protein synthesis. Pharmacol Rev. 16:223-43,Sep 1964.
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
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 481


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Parameters
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