Difference between revisions of "Part:BBa K1934061"
m
 
Line 15: Line 15:
 
<html>
 
<html>
 
<h3 id="RT">1. Overproduction and purification of the p66 protein subunit</h3>
 
<h3 id="RT">1. Overproduction and purification of the p66 protein subunit</h3>
−
<p>The BBa_K1934061 part conceived by the 2016 INSA-Lyon team and synthesized by Genecust was cloned into pUC57 and transformed into the <i>E. 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 p66 was purified on Ni-NTA columns from Qiagen (the protocol at the following <a href="https://www.qiagen.com/us/resources/resourcedetail?id=3fc8c76d-6d21-4887-9bf8-f35f78fcc2f2&lang=en">link)</a>. Samples at different steps of the process were analyzed on a SDS-PAGE gel 12% and protein revealed by staining with Coomassie Blue. P66 represent 22% of the total protein produced by the recombinant NM522/pUC57- BBa_K1934061 strain (See figure below, lane 1). Purification on Ni-NTA of the poly-His tagged p66 allows to obtain this subunit with a purification level of 87%.  </p>  
+
<p>The BBa_K1934061 part conceived by the 2016 INSA-Lyon team and synthesized by Genecust was cloned into pUC57 and transformed into the <i>E. 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 p66 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. p66 represent 22% of the total protein produced by the recombinant NM522/pUC57- BBa_K1934061 strain (See figure below, lane 1). Purification on Ni-NTA of the poly-His tagged p66 allows to obtain this subunit with a purification level of 87%.  </p>  
  
−
<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/3/38/INSA-Lyon_p66.jpg" width = "400"/><figcaption><b>Figure 1. Overproduction and purification of the p66 protein subunit </b> Lane 1 shows the crude cellular extract with a major band migrating at approximately 64 kDa. Such a band could is visible but attenuated in the different washing steps (lanes 2, 3, 4 and 5). Lane 6 show the purified recovered protein. The elution allows to recover 64% of the overproduced p66. P66 is pure in this fraction at 87% </figcaption></figure>
+
<figure><p style="text-align:center;"><img src="https://static.igem.org/mediawiki/parts/3/38/INSA-Lyon_p66.jpg" width = "400"/><figcaption><b>Figure 1. Overproduction and purification of the p66 protein subunit </b> Lane 1 shows the crude cellular extract with a major band migrating at approximately 64 kDa. Such a band could is visible but attenuated in the different washing steps (lanes 2, 3, 4 and 5). Lane 6 show the purified recovered protein. The elution allows to recover 64% of the overproduced p66. Protein is pure in this fraction at 87% </figcaption></figure>
  
 
<h3 id="RT">2. Assembly of a functional HIV-1 reverse transcriptase</h3>
 
<h3 id="RT">2. Assembly of a functional HIV-1 reverse transcriptase</h3>

Latest revision as of 19:39, 25 October 2016


p66 subunit of HIV reverse transcriptase

This part contains the sequence of the p66 subunit of the HIV reverse transcriptase. It is not functional on its own and must be associated with the p51 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 238
    Illegal BglII site found at 937
    Illegal BglII site found at 1100
    Illegal BglII site found at 1150
    Illegal XhoI site found at 1800
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 1739
    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 p66 protein subunit

The BBa_K1934061 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 p66 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. p66 represent 22% of the total protein produced by the recombinant NM522/pUC57- BBa_K1934061 strain (See figure below, lane 1). Purification on Ni-NTA of the poly-His tagged p66 allows to obtain this subunit with a purification level of 87%.

Figure 1. Overproduction and purification of the p66 protein subunit Lane 1 shows the crude cellular extract with a major band migrating at approximately 64 kDa. Such a band could is visible but attenuated in the different washing steps (lanes 2, 3, 4 and 5). Lane 6 show the purified recovered protein. The elution allows to recover 64% of the overproduced p66. Protein is pure in this fraction at 87%

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. p66, produced and purified as described above, was mixed in PBS in equimolar ratio with p51, produced and purified from BBa K1934060. 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.