Difference between revisions of "Part:BBa K3815034"
 
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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 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 p66. The genes of interest were cloned into pET-11a. <br>
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[[File:Detection_RT.png|300px|thumb|right|Fig1. From left to right: 1-4:  newly prepared homemade RT with different enzyme concentrations, 5: no enzyme,  6: Bst3.0, 7: old homemade RT,  8: no template control]]
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This part is used to purify 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 p66.   We examined our homemade reverse transcriptase activity in vitro. We used total yeast DNA/RNA for this assay. We selected the intron-containing region of Rpl19A as a template for reverse transcription. If there is no reverse transcription, the genomic DNA including an intron is amplified by PCR, resulting in a band of 876 bp. On the other hand, if cDNA is correctly synthesized by reverse transcription, a band of 370 bp without an intron should appear. As shown below, the cDNA band was successfully observed in an RT-dependent manner. From this result, we concluded the activity of homemade RT.
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<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.
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</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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Fig2 shows the result of SDS-PAGE.
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The lane 1,2,3,4 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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Lane 1,2,3,4 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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==Reference==
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Kellner, M.J., Ross, J.J., Schnabl, J., Dekens, M.P.S., Heinen, R., Grishkovskaya, I., Bauer, B., Stadlmann, J., Menéndez-Arias, L., Fritsche-Polanz, R., et al. (2020). A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing. <br>
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<!-- Uncomment this to enable Functional Parameter display  
 
<!-- Uncomment this to enable Functional Parameter display  
 
===Functional Parameters===
 
===Functional Parameters===
 
<partinfo>BBa_K3815034 parameters</partinfo>
 
<partinfo>BBa_K3815034 parameters</partinfo>
 
<!-- —>
 
<!-- —>
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==Reference==
 
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https://www.biorxiv.org/content/10.1101/2020.06.23.166397v2.full.pdf
 

Latest revision as of 02:25, 22 October 2021



HIV-1 reverse transcriptase p66

Description of this part

Targeted protein

Fig1. From left to right: 1-4: newly prepared homemade RT with different enzyme concentrations, 5: no enzyme, 6: Bst3.0, 7: old homemade RT, 8: no template control

This part is used to purify 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 p66. We examined our homemade reverse transcriptase activity in vitro. We used total yeast DNA/RNA for this assay. We selected the intron-containing region of Rpl19A as a template for reverse transcription. If there is no reverse transcription, the genomic DNA including an intron is amplified by PCR, resulting in a band of 876 bp. On the other hand, if cDNA is correctly synthesized by reverse transcription, a band of 370 bp without an intron should appear. As shown below, the cDNA band was successfully observed in an RT-dependent manner. From this result, we concluded the activity of homemade RT.



Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal PstI site found at 320
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal PstI site found at 320
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal PstI site found at 320
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal PstI site found at 320
    Illegal AgeI site found at 966
  • 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.
  • 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.(70mM, 95mM, 120mM, 270mM)

Fig2 shows the result of SDS-PAGE. Lane 1,2,3,4 are the result of Reverse Transcriptase.
We observed the correct bands in the SDS-PAGE gel. The arrowheads indicate the targeted proteins.

Reference

Kellner, M.J., Ross, J.J., Schnabl, J., Dekens, M.P.S., Heinen, R., Grishkovskaya, I., Bauer, B., Stadlmann, J., Menéndez-Arias, L., Fritsche-Polanz, R., et al. (2020). A rapid, highly sensitive and open-access SARS-CoV-2 detection assay for laboratory and home testing.