Difference between revisions of "Team:Freiburg/Project/pRIG15 17"

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We inserted the sequence coding for HIV tat/pol/gag/env into pET_22b+ for overexpression in <em>E.coli</em>. We could show interaction of the HIV tat/pol/gag/env antigen with a polyclonal anti-HIV-1 P24 antibody (Fig. 2)
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We inserted the sequence coding for HIV tat/pol/gag/env into pET_22b+ for overexpression in <em>E.coli</em>. We could show interaction of the HIV tat/pol/gag/env antigen with a polyclonal anti-HIV-1 p24 antibody (Fig. 2)
 
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                       <p><strong>Figure 2: Western Blot of <a href="http://parts.igem.org/Part:BBa_K1621004">HIV multi-epitopic antigen</a>.</strong> HIV multi-epitopic antigen was analyzed by 12,5% SDS-PAGE. The anti-HIV-1 P24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa.</p>
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                       <p><strong>Figure 2: Western Blot of <a href="http://parts.igem.org/Part:BBa_K1621004">HIV multi-epitopic antigen</a>.</strong> HIV multi-epitopic antigen was analyzed by 12.5% SDS-PAGE. The anti-HIV-1 p24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa.</p>
 
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Get the sequence in GeneBank format: <a class="media" href="https://static.igem.org/mediawiki/2015/e/e6/Freiburg_2015_BBa_K1621004.gb" title="2015_Freiburg_BBa_K1621004" src="https://static.igem.org/mediawiki/2015/e/e6/Freiburg_2015_BBa_K1621004.gb">BBa_K1621004.gb</a>.
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Link to GeneBank file: <a class="media" href="https://static.igem.org/mediawiki/2015/e/e6/Freiburg_2015_BBa_K1621004.gb" title="2015_Freiburg_BBa_K1621004" src="https://static.igem.org/mediawiki/2015/e/e6/Freiburg_2015_BBa_K1621004.gb">BBa_K1621004.gb</a>.
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Link to Registry: <a href="http://parts.igem.org/Parts:BBa_K1621004" target="_blank">BBa_K1621004</a>
 
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Revision as of 16:44, 16 September 2015

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pRIG15_17

The Human Immunodeficiency Virus (HIV) causes acquired immunodeficiency syndrome, AIDS, which leads to major impairments of the immunsystem. 1) For our experiments we used a sequence coding for a recombinant protein that contains six epitopes from different proteins of the HI Virus (HIV-1-trans-activating (tat) encoding region, one epitope of the reverse transcriptase, one of the p24 protein, one of the envelope protein gp41, one of gp120).2)

Figure 1: pRIG15_17. BBa_K1621004 inserted into the submission backbone pSB1C3.

To insert the sequence for HIV tat/pol/gag/env into pSB1C3 we designed Gibson primers with compatible overhangs that also included the start codon ATG. This fragment was amplified via PCR (Link zum Labjournal-Eintrag) and then assembled with the digested pSB1C3 backbone using Gibson assembly. To prove correct insertion of our fragment we did a test digest and sent the whole plasmid for sequencing.

We inserted the sequence coding for HIV tat/pol/gag/env into pET_22b+ for overexpression in E.coli. We could show interaction of the HIV tat/pol/gag/env antigen with a polyclonal anti-HIV-1 p24 antibody (Fig. 2)

Figure 2: Western Blot of HIV multi-epitopic antigen. HIV multi-epitopic antigen was analyzed by 12.5% SDS-PAGE. The anti-HIV-1 p24 polyclonal antibody was used in a dilution of 1:5000. The secondary antibody (anti-rabbit HRP) was diluted 1:5000. The expected molecular weigth is 20.5 kDa.





Link to GeneBank file: BBa_K1621004.gb.
Link to Registry: BBa_K1621004