Difference between revisions of "Part:BBa K3815032"
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==Purification== | ==Purification== | ||
| + | [[File:Detection_SDS-PAGE_gel.png|300px|thumb|right|Fig1. SDS-PAGE of purified proteins ]] | ||
<h3><font size="4.5">Expression</font> </h3> | <h3><font size="4.5">Expression</font> </h3> | ||
<ul> | <ul> | ||
Revision as of 16:33, 21 October 2021
B. stearothermophilus DNA polymerase
Description of this part
Targeted protein
This part is used to purify your own homemade BST DNA polymerase. This is derived from Geobacillus stearothermophilus. This enzyme is used for strand-displacing DNA synthesis in LAMP(Loop-Mediated Isothermal Amplification) or RT-LAMP(Reverse Transcription Loop-Mediated Isothermal Amplification). The enzyme has 5′-3′ polymerase activity and strand displacement activity, but it lacks 3′-5′ exonuclease activity. It also has reverse transcription activity.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 393
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1003
- 1000COMPATIBLE WITH RFC[1000]
Purification
Expression
- Cells were cultivated in 1000ml LB media at 37oC shaking at 180 rpm to the log-phase.
- when the OD600 reaches 0.35-0.7, IPTG was added at the final concentration of 0.5mM to induce the peptide expression.
- After 3 hours of induction, cells were harvested and frozen in liquid-N2.
Purification
After the cell disruption by sonication, each protein was purified using Ni-NTA beads. Samples were eluted by a four-step gradient of Imidazole(70mM, 95mM, 120mM, 270mM).
Fig1 shows the result of SDS-PAGE.
The lane 9,10,11,12 are the result of BST DNA polymerase.
We observed the correct bands in the SDS-PAGE gel. The arrowheads indicate the targeted proteins.