Part:BBa_K3771007

CoaBC-6xHis

Description

CoaBC is an enzyme that weighs 55.4 kDa. CoaBC functions in the JJU10-CoaBC taurine biosynthesis pathway, converting L-cystate to taurine. [3]

Usage

The three taurine production pathways incorporated into our E. coli include the L-cysteine sulfinic acid pathway, L-cysteine sulfonic acid pathway, and the JJU-CoaBC pathway[1,2].

CoaBC enzyme was used in vitro testing of taurine production. The sequence for CoaBC enzyme and trc promoter were ligated and transformed into E. coli to calculate taurine production using high-performance liquid chromatography (HPLC).

Figure.1 Taurine pathways in E. coli [1,2].

CoaBC is an enzyme in the JJU10-CoaBC pathway, one of three possible taurine synthesis pathways. Its main function is to convert L-cystate to taurine.

Characterization

The CoaBC fragment was synthesized by IDT and amplified by PCR. Agarose gel electrophoresis result is shown in Fig. 2.

Figure. 2 Confirmation of coaBC fragment by PCR. M: Marker; Lane 1: coaBC (1221 bp)

SDS-PAGE of the CoaBC enzyme to confirm protein expression.

Fig. 3 Confirmation of protein expression of CoaBC.M: Marker; Lane1: whole cell of CoaBC in BL21(DE3); Lane2: soluble protein of CoaBC in BL21(DE3) (~45 kDa)

Taurine production yield of CoaBC with other production enzymes calculated by high-performance liquid chromatography (HPLC).

Fig. 4 Taurine production of JJU in BL21(DE3) and CoaBC in BL21(DE3) in supernatant and whole cell samples.

References

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 NgoMIV site found at 583
  
• 1000
  COMPATIBLE WITH RFC[1000]