Part:BBa_K4171010

PlacI-cysK

Background

P_lacI-cysK is constructed to express CysK (BBa_K4171000), an enzyme involved in cysteine synthesis pathway that catalyzes O-acytel-serine (OAS) and sulfide into cysteine.

Usage

P_lacI-cysK-cysE* (BBa_K4171012) was cloned to convert OAS and selenide into selenocysteine, for the similarities shared between sulfide and selenide. Therefore, we first cloned P_lacI-cysK (BBa_K4171010) and sent it to the E. coli △cysK mutant with dysfunctional CysK, to confirm the function of CysK.

Fig. 2. In vivo Sec synthesis pathway

Characterization

Fig. 3. Confirmation of P_lacI-cysK by colony PCR. M: Marker; Lane 1-3: different colonies of BBa_K4171010 (2360bp).

To evaluate CysK function, we transformed BBa_K4171010 into E. coli △cysK mutant and observed its growth curve. Theoretically, once we conducted transformation successfully, the plasmid should compensate for the mutation, which is corresponding to the results shown below.

Fig. 2. Confirmation of CysK function by transforming P_lacI-cysK (BBa_K4171010) into E. coli △cysK mutant.

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
  
• 1000
  COMPATIBLE WITH RFC[1000]