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Is it possible for a gene disrupted by an antibiotic resistance...
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Matthew Leib
Is it possible for a gene disrupted by an antibiotic resistance...
If the downstream ORF is fused in frame with the hyg resistance gene, it is quite possible that the resulting fusion protein may retain some activity, albeit probably not as strong as the original protein. This occurs not infrequently with vectors including a lacZ∆M15 gene for blue/white selection; with some clonings, the fusion results in pale blue colonies.
If the downstream ORF is fused in frame with the hyg resistance gene, it is quite possible that the resulting fusion protein may retain some activity, albeit probably not as strong as the original protein. This occurs not infrequently with vectors including a lacZ∆M15 gene for blue/white selection; with some clonings, the fusion results in pale blue colonies.
Seems like it could definitely be possible but it would still remain to be seen if the 5' fusion of the hygromycin resistance gene has negative effects on transcript regulation or splicing. If that proceeds successfully, post-translational events are still an open question because the presence of a fusion protein can inhibit the native protein sequence from folding correctly or, even if it does, maintaining its function.
If the goal is to knock out gene function, I think you'll probably want to set up the guide sequences and homology arms so that the homology-directed repair puts the hygromycin resistance gene in in the wrong frame...just to be sure.
Seems like it could definitely be possible but it would still remain to be seen if the 5' fusion of the hygromycin resistance gene has negative effects on transcript regulation or splicing. If that proceeds successfully, post-translational events are still an open question because the presence of a fusion protein can inhibit the native protein sequence from folding correctly or, even if it does, maintaining its function.
If the goal is to knock out gene function, I think you'll probably want to set up the guide sequences and homology arms so that the homology-directed repair puts the hygromycin resistance gene in in the wrong frame...just to be sure.
If the downstream ORF is fused in frame with the hyg resistance gene, it is quite possible that the resulting fusion protein may retain some activity, albeit probably not as strong as the original protein. This occurs not infrequently with vectors including a lacZ∆M15 gene for blue/white selection; with some clonings, the fusion results in pale blue colonies.
If the downstream ORF is fused in frame with the hyg resistance gene, it is quite possible that the resulting fusion protein may retain some activity, albeit probably not as strong as the original protein. This occurs not infrequently with vectors including a lacZ∆M15 gene for blue/white selection; with some clonings, the fusion results in pale blue colonies.
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I think it is, (you surely get the antibiotic). I'm not sure on complementation or the "deleterious" effect be fixed.
I think it is, (you surely get the antibiotic). I'm not sure on complementation or the "deleterious" effect be fixed.
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Seems like it could definitely be possible but it would still remain to be seen if the 5' fusion of the hygromycin resistance gene has negative effects on transcript regulation or splicing. If that proceeds successfully, post-translational events are still an open question because the presence of a fusion protein can inhibit the native protein sequence from folding correctly or, even if it does, maintaining its function.
If the goal is to knock out gene function, I think you'll probably want to set up the guide sequences and homology arms so that the homology-directed repair puts the hygromycin resistance gene in in the wrong frame...just to be sure.
Seems like it could definitely be possible but it would still remain to be seen if the 5' fusion of the hygromycin resistance gene has negative effects on transcript regulation or splicing. If that proceeds successfully, post-translational events are still an open question because the presence of a fusion protein can inhibit the native protein sequence from folding correctly or, even if it does, maintaining its function.
If the goal is to knock out gene function, I think you'll probably want to set up the guide sequences and homology arms so that the homology-directed repair puts the hygromycin resistance gene in in the wrong frame...just to be sure.
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