Part:BBa_K4171005

YdeD

Background

YdeD (BBa_K4171005) is a transmembrane protein that pumps cysteine (Cys) and other metabolic molecules out of the cells.

Usage

YdeD (BBa_K4171005) was used to collaborate with P_lacI-cysK-cysE* (BBa_K4171012) to yield more selenocysteine (Sec).

Excessive Sec that accumulates inside the cells has negative effects on the growth of bacteria. As a result, the total production of Sec is likely to decrease due to the reduced cell numbers. Besides, in order to extract Sec, we have to disrupt the cells and break the cell membranes, which instead makes it difficult to separate Sec from other amino acids and disrupted cells.

To solve these problems, we added ydeD into this pathway^[1]. With YdeD (BBa_K4171005), E. coli automatically pumps out Sec, making the cell a natural factory of Sec synthesis.

Fig. 1. In vivo Sec synthesis pathway

Characterization

Fig. 2. Confirmation of ydeD (BBa_K4171005) by PCR. M: Marker; Lane 1: ydeD (919bp).

When expressed together with P_lacI-cysK-cysE* in E. coli, YdeD (BBa_K4171005) functions to support the lives of microorganisms. To yield Sec, sodium selenite was added as one of the substrates, and part of it was reduced to selenium by bacteria, which made the medium red. Surprisingly, with YdeD overexpression, the color of the medium was obviously lighter. To confirm the hypothesis that YdeD promotes higher Sec production and thus makes less sodium selenite being transformed into selenium, CFU was conducted to see whether YdeD helps bacteria survive under the toxicity of selenide.

Based on the result (Fig. 3), only the cell numbers of groups 7 and 8 showed obvious differences, which proves two things. First, sodium selenite and its metabolites contributed to the difference in the survival of E. coli. Second, the function of YdeD (BBa_K4171005) was not that obvious without CysK and CysE* in groups 3 and 4, since Sec was not synthesized as much as those in 7 and 8. Combining this information, we successfully evaluated YdeD function.

Fig. 3. CFU examination of YdeD (BBa_K4171005) function.

Reference

[1] Wiriyathanawudhiwong N, Ohtsu I, Li ZD, Mori H, Takagi H. The outer membrane TolC is involved in cysteine tolerance and overproduction in Escherichia coli. Appl Microbiol Biotechnol. 2009;81(5):903-913. doi:10.1007/s00253-008-1686-9 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]