Difference between revisions of "Part:BBa K4195085"

Latest revision as of 16:42, 10 October 2022

Gam-his

Biology

Gam Nuclease Inhibitor is a recombinant protein that inhibits Exonuclease V (RecBCD) activity and stabilizes linear DNA templates in E. coli based in vitro protein synthesis reactions (1). While protecting linear DNA, it does not significantly affect expression efficiency.

Usage and Design

His-tag (6×His) is expressed at the N end of Gam for protein purification. We assembled BBa_K3222000 and BBa_B0034 with this part into the vector pET-28a(+), and the constructed plasmids were transformed into E. coli BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing. Our cell-free expression system uses linear DNA as a template for protein expression. In the cell-free expression system, Gam is added to protect linear DNA.

Characterization

1. Agarose Gel Electrophoresis

After constructing the part on the expression vector pET-28a(+), the plasmid is transferred into E. coli BL21(DE3), then the positive transformants are selected by kanamycin and confirmed by colony PCR and sequencing.

Fig. 1 The result of colony PCR. Plasmid.

2. SDS-PAGE

The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3). After being cultivated and induced by ITPG, GE AKTA Prime Plus FPLC System is employed to get purified protein from broken supernatant. Purified protein is verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining.

Fig. 2 SDS-PAGE analysis of protein in lysate of E. coli BL21(DE3) and the eluant.

Reference

1. A. Didovyk, T. Tonooka, L. Tsimring, J. Hasty, Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. ACS Synth Biol 6, 2198-2208 (2017).

Sequence and Features