Part:BBa_K4171012

PlacI-cysK-cysE*

Background

P_lacI-cysK-cysE* is constructed to express CysK and CysE*. Both CysK (BBa_K4171000) and CysE* (BBa_K4171004) are engaged in cysteine (Cys) synthesis pathway in E. coli. CysE* catalyzes serine (Ser) into O-acetyl-serine (OAS) while CysK turns OAS and sulfide into cysteine.

Usage

E. coli can produce Cys on its own if given Ser and sulfide sources like sulfate, sulfite, sulfide, etc. According to studies. Cys and Sec have been proven to share a lot of similarities^[1]. This gave us the idea of replacing sulfur with selenium and producing selenocysteine (Sec) by mimicking the natural Cys synthesis pathway. Therefore, P_lacI-cysK-cysE* (BBa_K4171012) is constructed to confirm whether the enzymes that originally produce Cys can also synthesize Sec as well with the present selenide instead of sulfide.

Fig. 1. In vivo Sec synthesis pathway

Characterization

Fig. 2. Confirmation of P_lacI-cysK-cysE* (BBa_K4171012) by colony PCR. M: Marker; Lane 1: P_lacI-cysK-cysE* (2259bp).

To evaluate CysE* function, we transformed BBa_K4171012 into E. coli △cysE mutant and observed its growth curve. Theoretically, once we conducted transformation successfully, the plasmid should compensate for the mutant, which is corresponding to the result shown below.

Fig. 3. Confirmation of CysE* function by transforming P_lacI-cysK-cysE* (BBa_K4171012) into E. coli △cysE mutant.

Reference

[1]Ng, B. and Anderson, J., 1978. Synthesis of selenocysteine by cysteine synthases from selenium accumulator and non-accumulator plants. Phytochemistry, 17(12), pp.2069-2074.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 695
  Illegal AgeI site found at 1896
  
• 1000
  COMPATIBLE WITH RFC[1000]