![](https://parts.igem.org/images/partbypart/icon_coding.png)
Part:BBa_K4171001
SerRS
Background
SerRS is an aminoacyl-tRNA synthase weighing around 4.8kDa. It catalyzes the aminoacylation of tRNASec with serine to form L-seryl-tRNASer, a substrate that can be later converted to L-selenocysteinyl-tRNASec.
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 tRNASer (BBa_K4171007), tRNASec (BBa_K4171008), and tRNAUTuX (BBa_K4171009) with serine.
![](https://static.igem.wiki/teams/4171/wiki/parts/in-vitro-pathway.png)
Fig. 1. In vitro Sec synthesis pathway
Characterization
SDS-PAGE analysis was conducted to confirm the success of SerRS purification.
![](https://static.igem.wiki/teams/4171/wiki/parts/serrs-sds-page-parts.png)
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.
![](https://static.igem.wiki/teams/4171/wiki/parts/aminoacylation-urea.jpg)
Fig. 3. Confirmation of the result after aminoacylation by Urea PAGE. Lane1: tRNASer; Lane2: seryl-tRNASer; Lane3: tRNASec; Lane4: seryl-tRNASec; Lane5: tRNAUTuX; Lane6: seryl-tRNAUTuX
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1064
Illegal BamHI site found at 1037 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI.rc site found at 100
Illegal SapI.rc site found at 238
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