Difference between revisions of "Part:BBa K1934060"
m |
m |
||
| Line 15: | Line 15: | ||
<html> | <html> | ||
<h3 id="RT">1. Overproduction and purification of the p51 protein subunit</h3> | <h3 id="RT">1. Overproduction and purification of the p51 protein subunit</h3> | ||
| − | <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. | + | <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> |
| − | <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. | + | <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> |
<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 19:40, 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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 1100
Illegal BglII site found at 1150
Illegal XhoI site found at 1419 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 1067
Illegal AgeI site found at 1182 - 1000INCOMPATIBLE 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%.
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).