Difference between revisions of "Part:BBa K3815033"

 
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<partinfo>BBa_K3815033 short</partinfo>
 
<partinfo>BBa_K3815033 short</partinfo>
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==Description of this part==
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<h3><font size="3">Targeted protein</font> </h3>
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This part is used to purify your own homemade Reverse Transcriptase. This is derived from HIV-1(Human Immunodeficiency Virus). This enzyme is more thermotolerant than normal reverse transcriptase, making it useful for LAMP(Loop-Mediated Isothermal Amplification)) and RT-LAMP(Reverse Transcription Loop-Mediated Isothermal Amplification). A peptide made from one ORF is partially digested in an infected cell and becomes two fragments, p66 and p51. It is known that the HIV-1 RT functions when the p66 and p51 form a heterodimer. To purify them as recombinant proteins in E.coli, we separately expressed and purified p66 and p51, and dimerized them in vitro before use. This part is to express p51. The genes of interest were cloned into pET-11a. <br>
  
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
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<partinfo>BBa_K3815033 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K3815032 SequenceAndFeatures</partinfo>
  
  
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==Purification==
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<h3><font size="4.5">Expression</font> </h3>
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<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>when the OD600 reaches 0.35-0.7, IPTG was added at the final concentration of 0.5mM to induce the peptide expression.
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<li>After 3 hours of induction, cells were harvested and frozen in liquid-N2.
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<li>We added purified p66(BBa_K3815034) and p51(BBa_K3815033) to the same tube and incubated them at -30<sup>o</sup>C for 4 days to make a dimer.
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</ul>
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<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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<br>
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Fig1 shows the result of SDS-PAGE.
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The lane 5,6,7,8 are the result of Reverse Transcriptase.<br> We observed the correct bands in the SDS-PAGE gel. The arrowheads indicate the targeted proteins.
 
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===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3815033 parameters</partinfo>
 
<partinfo>BBa_K3815033 parameters</partinfo>
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==Reference==
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https://www.biorxiv.org/content/10.1101/2020.06.23.166397v2.full.pdf

Revision as of 13:34, 21 October 2021


HIV-1 reverse transcriptase p51

Description of this part

Targeted protein

This part is used to purify your own homemade Reverse Transcriptase. This is derived from HIV-1(Human Immunodeficiency Virus). This enzyme is more thermotolerant than normal reverse transcriptase, making it useful for LAMP(Loop-Mediated Isothermal Amplification)) and RT-LAMP(Reverse Transcription Loop-Mediated Isothermal Amplification). A peptide made from one ORF is partially digested in an infected cell and becomes two fragments, p66 and p51. It is known that the HIV-1 RT functions when the p66 and p51 form a heterodimer. To purify them as recombinant proteins in E.coli, we separately expressed and purified p66 and p51, and dimerized them in vitro before use. This part is to express p51. The genes of interest were cloned into pET-11a.


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

Expression

  • Cells were grown 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.
  • We added purified p66(BBa_K3815034) and p51(BBa_K3815033) to the same tube and incubated them at -30oC for 4 days to make a dimer.

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.

Fig1 shows the result of SDS-PAGE. The lane 5,6,7,8 are the result of Reverse Transcriptase.
We observed the correct bands in the SDS-PAGE gel. The arrowheads indicate the targeted proteins.