Difference between revisions of "Part:BBa K1934060"
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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. Overproducing and purifying the part</h3>
 
<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>
 
<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>
  

Revision as of 15:54, 16 October 2016


p51 subunit of HIV reverse transcriptase

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.

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. Overproducing and purifying the part

This part was cloned into e. coli 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 page. The protein purification yield was 350% and the global recovery yield was 24 %.

Figure 1. Steps of purification of the p51 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.

2. Testing the reverse transcriptase

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.

Figure 2. RT-PCR with the purified HIV RT 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.
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.