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It has been suggested that bacteria transformed with pBlueScript vector, containing an insert in the middle of the lacZ' gene, can still give blue colour on X-gal, if the insert is small and ligated in-frame with the lacZ' gene (source: my course, ResearchGate).

I don't understand how it is possible that translation doesn't end at the stop codon encoded by the insert. Is this something to do with leaky termination? Moreover, even if leaky termination was possible, this would mean that there is ALWAYS a 1 in 3 chance of the insert being in-frame.

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  • $\begingroup$ Transcription doesn't stop at the stop codon. $\endgroup$ – canadianer Apr 12 '17 at 17:51
  • $\begingroup$ @canadianer Thanks! But why does it not? And also, if it doesn't stop at the stop codon, doesn't this mean that in 1 out of 3 cases, an insert would be ligated in-frame and the assay wouldn't work? $\endgroup$ – GingerBadger Apr 12 '17 at 18:09
  • $\begingroup$ Transcription has nothing to do with codons. Perhaps you meant translation? $\endgroup$ – canadianer Apr 12 '17 at 19:04
  • $\begingroup$ @canadianer Oops, this is awkward! Yes, this is what I meant! $\endgroup$ – GingerBadger Apr 12 '17 at 19:10
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Most inserts will disrupt beta-gal expression (by shifting the reading frame and introducing stop codons) and therefore remove the enzymatic activity required to cleave X-gal and produce the blue color.

Your source is agreeing with you - inserting a stop codon will stop translation (not transcription) and keep the white color.

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  • $\begingroup$ Thanks! But the source also says that "In rare cases, when the insert is very small and does not disrupt the reading frame, beta-galactosidase activity will occur even in the recombinant clones, so blue-white screening becomes irrelevant." Am I interpreting this incorrectly? $\endgroup$ – GingerBadger Apr 12 '17 at 18:08
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    $\begingroup$ I'm understanding the implication that, to still have beta-galactosidase activity, the insert needs to not have a stop codon. I think this is reasonable because the stop codon case is explicitly addressed in the previous paragraph. $\endgroup$ – mxwsn Apr 12 '17 at 18:11
  • $\begingroup$ But doesn't this mean that without a stop codon blue color will be produced in 1 in 3 cases (probability of in-frame ligation) and the assay is kind of pointless, so why would anyone use it like that? $\endgroup$ – GingerBadger Apr 12 '17 at 18:15
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    $\begingroup$ The insert needs to not disrupt protein folding on top of being small, not having a stop codon, and being in frame. $\endgroup$ – mxwsn Apr 12 '17 at 18:31
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    $\begingroup$ @mxwsn You should add this to your answer. $\endgroup$ – canadianer Apr 12 '17 at 19:45

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