Difference between revisions of "Part:BBa K3815032"
 
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<h3><font size="3">Targeted protein</font> </h3>
 
<h3><font size="3">Targeted protein</font> </h3>
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This part is used to purify your own homemade BST DNA polymerase. This is derived from <i>Geobacillus stearothermophilus</i>. 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. The genes of interest were cloned into pET-11a. <br>
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[[File:Detection_DNA_polymerase_LAMP.png|300px|thumb|right|Fig1. ladder-like bands that are typical of LAMP reactions ]]
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This part is used to purify homemade BST DNA polymerase. This is derived from <i>Geobacillus stearothermophilus</i>. 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. In our experiments, we observed the activity of homemade DNA polymerase, which is comparable to commercial sources (BST3.0 from NEB) in LAMP reaction against DMV(dahlia mosaic virus). In Fig. 1 you see ladder-like bands that are typical of LAMP reactions. <br>
  
  
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==Purification==
 
==Purification==
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[[File:Detection_SDS-PAGE_gel.png|300px|thumb|right|Fig2. SDS-PAGE of purified proteins ]]
 
<h3><font size="4.5">Expression</font> </h3>
 
<h3><font size="4.5">Expression</font> </h3>
 
<ul>
 
<ul>
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<li>Cells were grown in 1000ml LB media at 37<sup>o</sup>C shaking at 180 rpm to the log-phase.
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<li>Cells were cultivated in 1000ml LB media at 37<sup>o</sup>C shaking at 180 rpm to the log-phase.
 
<li>when the OD600 reaches 0.35-0.7, IPTG was added at the final concentration of 0.5mM to induce the peptide expression.
 
<li>when the OD600 reaches 0.35-0.7, IPTG was added at the final concentration of 0.5mM to induce the peptide expression.
 
<li>After 3 hours of induction, cells were harvested and frozen in liquid-N2.
 
<li>After 3 hours of induction, cells were harvested and frozen in liquid-N2.
 
</ul>
 
</ul>
 
<h3><font size="4.5">Purification </font></h3>
 
<h3><font size="4.5">Purification </font></h3>
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After the cell disruption by sonication, each protein was purified using Ni-NTA beads. Samples were eluted by a four-step gradient of Imidazole. <br>
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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). <br>
 
<br>
 
<br>
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Fig1 shows the result of SDS-PAGE.
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Fig. 2 shows the result of SDS-PAGE.
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The lane 9,10,11,12 are the result of BST DNA polymerase.<br> We observed the correct bands in the SDS-PAGE gel. The arrowheads indicate the targeted proteins.
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Lane 9,10,11,12 are the result of BST DNA polymerase.<br> We observed the correct bands in the SDS-PAGE gel. The arrowheads indicate the targeted proteins.
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<br>
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==Reference==
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https://www.lucigen.com/docs/manuals/MA073-Bst-DNA-Polymerase-8-U.pdf
 
<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
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<br>
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3815032 parameters</partinfo>
 
<partinfo>BBa_K3815032 parameters</partinfo>
 
<!-- —>
 
<!-- —>
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==Reference==
 
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https://www.google.com/url?sa=t&rct=j&q=&esrc=s&source=web&cd=&cad=rja&uact=8&ved=2ahUKEwittMT9v9vzAhVEAIgKHcOCBZkQFnoECAwQAw&url=https%3A%2F%2Fwww.lucigen.com%2Fdocs%2Fmanuals%2FMA073-Bst-DNA-Polymerase-8-U.pdf&usg=AOvVaw0XhXw3ov-2dDNC_D2OT3Yt
 

Latest revision as of 02:23, 22 October 2021


B. stearothermophilus DNA polymerase

Description of this part

Targeted protein

Fig1. ladder-like bands that are typical of LAMP reactions

This part is used to purify 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. In our experiments, we observed the activity of homemade DNA polymerase, which is comparable to commercial sources (BST3.0 from NEB) in LAMP reaction against DMV(dahlia mosaic virus). In Fig. 1 you see ladder-like bands that are typical of LAMP reactions.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 393
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1003
  • 1000
    COMPATIBLE WITH RFC[1000]


Purification

Fig2. SDS-PAGE of purified proteins

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).

Fig. 2 shows the result of SDS-PAGE. 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.


Reference

https://www.lucigen.com/docs/manuals/MA073-Bst-DNA-Polymerase-8-U.pdf