Part:BBa_K4171001

SerRS

Background

SerRS is an aminoacyl-tRNA synthase weighing around 4.8kDa. It catalyzes the aminoacylation of tRNA^Sec with serine to form L-seryl-tRNA^Ser, a substrate that can be later converted to L-selenocysteinyl-tRNA^Sec.

Usage

SerRS (BBa_K4171001) was overexpressed in E. coli MG1655 and purified by His-Tag protein purification column. Then, purified SerRS was used to bind tRNA^Ser (BBa_K4171007), tRNA^Sec (BBa_K4171008), and tRNA^UTuX (BBa_K4171009) with serine.

Fig. 1. In vitro Sec synthesis pathway

Characterization

SDS-PAGE analysis was conducted to confirm the success of SerRS purification.

Fig. 2. Confirmation of SerRS (BBa_K4171001) purification by SDS-PAGE. M: Marker; Lane 1: SerRS before condensing (~48kDa); Lane2: SerRS after condensing.

Furthermore, Urea PAGE was performed to confirm the success of aminoacylation between tRNA and serine. The bands of seryl-tRNA have upshifted compared to the band of the tRNA. The result shows that the amino acid was successfully charged to the tRNA.

Fig. 3. Confirmation of the result after aminoacylation by Urea PAGE. Lane1: tRNA^Ser; Lane2: seryl-tRNA^Ser; Lane3: tRNA^Sec; Lane4: seryl-tRNA^Sec; Lane5: tRNA^UTuX; Lane6: seryl-tRNA^UTuX

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1064
  Illegal BamHI site found at 1037
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI.rc site found at 100
  Illegal SapI.rc site found at 238