Difference between revisions of "Part:BBa K4195085"
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<partinfo>BBa_K4195085 short</partinfo> | <partinfo>BBa_K4195085 short</partinfo> | ||
| − | + | ===Biology=== | |
| + | Gam Nuclease Inhibitor is a recombinant protein that inhibits Exonuclease V (RecBCD) activity and stabilizes linear DNA templates in <i>E. coli</i> based in vitro protein synthesis reactions(<i>1</i>). 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 <partinfo>BBa_K3222000</partinfo> and <partinfo>BBa_B0034</partinfo> with this part into the vector pET-28a(+), and the constructed plasmids were transformed into <i>E. coli</i> 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 <i>E. coli</i> BL21(DE3), then the positive transformants are selected by kanamycin and confirmed by colony PCR and sequencing. | ||
| + | ====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. | ||
| + | ===Reference=== | ||
| + | 1. A. Didovyk, T. Tonooka, L. Tsimring, J. Hasty, Rapid and Scalable Preparation of Bacterial Lysates for Cell-Free Gene Expression. <i>ACS Synth Biol</i> <b>6</b>, 2198-2208 (2017). | ||
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| + | |||
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Revision as of 15:48, 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.
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]