Difference between revisions of "Part:BBa K1934060"
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<partinfo>BBa_K1934060 short</partinfo>
 
<partinfo>BBa_K1934060 short</partinfo>
  
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This part contains the sequence of the p51 subunit of HIV reverse transcriptase. It is not functional on his own, to have an activity it must be associated with the p66 protein.  
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This part contains the sequence of the p51 subunit of the HIV reverse transcriptase. It is not functional on its own and must be associated with the p66 subunit to be functional.  
  
 
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=='''Characterization'''==
 
=='''Characterization'''==
 
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<h3 id="RT">1. Purifying the part</h3>
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<h3 id="RT">1. Overproduction and purification of the p51 protein subunit</h3>
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<p>This part was cloned into e. <i>coli</i> NM522. Then the bacterium were grown in LB overnight at 37°. We got an OD600 of 2.4 at this stage, enough for harvest. The p51 has been tagged with a polyHis tag. A NiNTA column is used to purify it. We used NiNTA columns kit from kiagen. The protocol may be downloaded on this <a href="url" https://www.qiagen.com/us/resources/resourcedetail?id=3fc8c76d-6d21-4887-9bf8-f35f78fcc2f2&lang=en> page</a>. The protein purification yield was 350% and the global recovery yield was 24 %.  </p>
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<p>The BBa_K1934060 part conceived by the 2016  INSA-Lyon team and synthesized by Genecust was cloned into pUC57 and transformed into the  E. <i>coli</i> NM522 strain. One recombinant clone was grown overnight in LB at 37°C. Cells were harvested and total proteins were extracted using lysozyme and benzonase lysis protocol. After centrifugation the supernatant was collected and used for purification. The poly-His tagged p51 was purified on Ni-NTA columns from Qiagen (protocol available <a href="https://www.qiagen.com/us/resources/resourcedetail?id=3fc8c76d-6d21-4887-9bf8-f35f78fcc2f2&lang=en">here</a>). Samples at different steps of the process were analyzed on a SDS-PAGE gel 12% and protein revealed by staining with Coomassie Blue. p51 represent 24% of the total protein produced by the recombinant NM522/pUC57- BBa_K1934060 strain (See figure below, lane A). Purification on Ni-NTA of the poly-His tagged p51 allows to obtain this subunit with a purification level of 84%.  </p>  
  
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/e/e9/INSA-Lyon_p51.png" width = "400"/><figcaption><b>Figure 1. Steps of purification of the p51 </b> Lane A shows the raw cellular extract. Lanes B,C,D and E shows the different wash step that were performed. On these lanes the p51 is not visible. Lanes F and G shows the purified recovered protein. </figcaption></figure>
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<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2016/e/e9/INSA-Lyon_p51.png" width = "400"/><figcaption><b>Figure 1. Overproduction and purification of the p51 protein subunit </b> Lane A shows the crude cellular extract with a major band migrating at approximately 52 kDa. Such a band could not be detected in the different washing steps (lanes B,C,D,E). Lanes F and G show the purified recovered protein. The first elution allows to recover 77% of the overproduced p51. Protein is pure in this fraction at 84% </figcaption></figure>
  
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<h3 id="RT">2. Testing the reverse transcriptase</h3>
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<h3 id="RT">2. Assembly of a functional HIV-1 reverse transcriptase</h3>
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Prior to any test p51 and p66 (BBa K1934061), were mixed together so as to get an equimolar mix, in PBS. At this stage both subunits are assembling together to form a functional HIV-1 RT. Then we ran a sigle step RT-PCR. The RNA used in this RT-PCR was the 16S fragment from the ribosomes. In a PCR tube a classic PCR mix was introduced we also added to this mixture the RT and the RNA template and both reverse and forward oligos. Then the mixture was reacted at 40° for 20 min. At this stage the RT transcribes the 16 RNA into cDNA. Then a classic PCR cycling was performed (35 cycles). The result was analyzed on an agarose gel.
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<figure><p style="https://static.igem.org/mediawiki/2016/0/00/INSA-Lyon_RT-PCR.png" width = "400"/><figcaption><b>Figure 2. RT-PCR with the purified HIV RT</b>  Lane A represent the total reaction mixture. A band is clearly visible, indicating that our RT has a correct activity. Lane B is the same mixture without RT and C is without Taq Polymerase. Without these enzymes the RT-PCR is inefficient and no bands are visible. Lane D is our control band, it was prepared by replacing RNA with its corresponding cDNA. Lane E is the same reaction mixture without the RNA template. Lane F was the same reaction but without the primers oligos.</figcaption></figure>
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The HIV-1 Reverse Transcriptase (RT) is a heterodimeric protein. Two subunits assemble to form the functional protein: the p51 and the p66 subunits.
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p51, produced and purified as described above, was mixed in PBS in equimolar ratio with p66, produced and purified from <a href="https://parts.igem.org/Part:BBa_K1934061">BBa_K1934061</a>. Assembly of a functional HIV-1 RT can be demonstrated by testing the enzymatic activity of the mix. Therefore, a single step RT-PCR was set, using the 16S RNA from the ribosomes as template and the ACG GCT ACC TTG TTA CGA CTT reverse and AGA GTT TGA TCC TGG CTC AG forward primers. Incubation at 40°C during 20 min enables the reverse transcription of the 16S RNA into cDNA. Then a classic PCR cycling was performed (35 cycles). The PCR mixture was then analyzed on an agarose gel (see below).  
  
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This experiment shows that our recombinant RT is fully functional. Plus it also shows that the p51 and the p66 fragments are able to assemble.  
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<figure><p style="text-align:center;"><img src= "https://static.igem.org/mediawiki/2016/0/00/INSA-Lyon_RT-PCR.png" width = "400"/><figcaption><b>Figure 2. A functional HIV-1 RT is generated from the assembly of p51 (BBa_K1934060) and p66 <a href="https://parts.igem.org/Part:BBa_K1934061">(BBa_K1934061)</a> protein subunits, previously overproduced and purified.</b> Analysis of the nucleic acids produced in the RT-PCR on agarose gel revealed under UV light after ethidium bromide staining. Lane A represents the total RT-PCR reaction mixture. A band migrating at 1200 bp that is absent on the controls is clearly visible in lane A. Controls - Lane B: same mixture without RT, lane C: without Taq Polymerase, lane D: replacing RNA with its corresponding cDNA, lane E: without the RNA template, Lane F: without primers.</figcaption></figure>
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These results indicate that the p51 and p66 protein subunits overproduced and purified from our DNA constructs can be assembled to form a functional RT.
  
 
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Latest revision as of 19:40, 25 October 2016


p51 subunit of HIV reverse transcriptase

This part contains the sequence of the p51 subunit of the HIV reverse transcriptase. It is not functional on its own and must be associated with the p66 subunit to be functional.

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 1100
    Illegal BglII site found at 1150
    Illegal XhoI site found at 1419
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 1067
    Illegal AgeI site found at 1182
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 20

Characterization

1. Overproduction and purification of the p51 protein subunit

The BBa_K1934060 part conceived by the 2016 INSA-Lyon team and synthesized by Genecust was cloned into pUC57 and transformed into the E. coli NM522 strain. One recombinant clone was grown overnight in LB at 37°C. Cells were harvested and total proteins were extracted using lysozyme and benzonase lysis protocol. After centrifugation the supernatant was collected and used for purification. The poly-His tagged p51 was purified on Ni-NTA columns from Qiagen (protocol available here). Samples at different steps of the process were analyzed on a SDS-PAGE gel 12% and protein revealed by staining with Coomassie Blue. p51 represent 24% of the total protein produced by the recombinant NM522/pUC57- BBa_K1934060 strain (See figure below, lane A). Purification on Ni-NTA of the poly-His tagged p51 allows to obtain this subunit with a purification level of 84%.

Figure 1. Overproduction and purification of the p51 protein subunit Lane A shows the crude cellular extract with a major band migrating at approximately 52 kDa. Such a band could not be detected in the different washing steps (lanes B,C,D,E). Lanes F and G show the purified recovered protein. The first elution allows to recover 77% of the overproduced p51. Protein is pure in this fraction at 84%

2. Assembly of a functional HIV-1 reverse transcriptase

The HIV-1 Reverse Transcriptase (RT) is a heterodimeric protein. Two subunits assemble to form the functional protein: the p51 and the p66 subunits. p51, produced and purified as described above, was mixed in PBS in equimolar ratio with p66, produced and purified from BBa_K1934061. Assembly of a functional HIV-1 RT can be demonstrated by testing the enzymatic activity of the mix. Therefore, a single step RT-PCR was set, using the 16S RNA from the ribosomes as template and the ACG GCT ACC TTG TTA CGA CTT reverse and AGA GTT TGA TCC TGG CTC AG forward primers. Incubation at 40°C during 20 min enables the reverse transcription of the 16S RNA into cDNA. Then a classic PCR cycling was performed (35 cycles). The PCR mixture was then analyzed on an agarose gel (see below).

Figure 2. A functional HIV-1 RT is generated from the assembly of p51 (BBa_K1934060) and p66 (BBa_K1934061) protein subunits, previously overproduced and purified. Analysis of the nucleic acids produced in the RT-PCR on agarose gel revealed under UV light after ethidium bromide staining. Lane A represents the total RT-PCR reaction mixture. A band migrating at 1200 bp that is absent on the controls is clearly visible in lane A. Controls - Lane B: same mixture without RT, lane C: without Taq Polymerase, lane D: replacing RNA with its corresponding cDNA, lane E: without the RNA template, Lane F: without primers.
These results indicate that the p51 and p66 protein subunits overproduced and purified from our DNA constructs can be assembled to form a functional RT.