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I'm considering working with the plasmid pRFHUE-eGFP and would like to replace the gpdA intron (which is the eGFP promoter region) with a promoter from another organism.

What would be a good strategy for replacing the intron if I already have the other gene in-hand? Would I cut out the fragment between the SalI and SmaI restriction sites and then insert the new promoter with compatible overhangs there?

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Your strategy would work, but if possible you might be better off using XmaI rather than SmaI, as the latter is a blunt-end cutting enzyme.

Furthermore, your strategy would not replace the entire gpdA promoter, if that's what you're after. You might wanna refer to their paper, where it's clear from Fig. 1 that the promoter region is much longer than the region between SalI and SmaI:

Fig 1. from Crespo-Sempere et al. (2011)

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    $\begingroup$ I've been appreciating your on-point synthetic biology answers! Want to support the proposal for a dedicated SynBio StackExchange site? area51.stackexchange.com/proposals/125068/synthetic-biology $\endgroup$
    – jakebeal
    Commented Apr 14, 2021 at 2:16
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    $\begingroup$ Thanks @jakebeal, but I'm of the humble opinion that Synthetic Biology questions should remain part of SE Biology. :) $\endgroup$
    – gaspanic
    Commented Apr 14, 2021 at 11:00
  • $\begingroup$ Your call, of course; my own reasoning is spelled out in area51.meta.stackexchange.com/a/32413/211190 $\endgroup$
    – jakebeal
    Commented Apr 14, 2021 at 11:20
  • $\begingroup$ Your answer has pointed out that the plasmid on AddGene and the one referenced in the paper are different. The directionality is reversed, they vary by 8bp in size and the annotations don't match. I wonder why this might be. $\endgroup$
    – doremi
    Commented Apr 14, 2021 at 16:03
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    $\begingroup$ Note that what you refer to as directionality here is irrelevant: one map is simply displayed as the reverse complement of the other. Second, annotations might differ because the authors chose a certain vocabulary for the paper, whereas the plasmid map you got from AddGene might be automatically annotated based on the DNA sequence (just guessing). And I wouldn't pay too much attention to the 8 bp difference in size. $\endgroup$
    – gaspanic
    Commented Apr 14, 2021 at 16:22

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