I am cloning an overexpression plasmid with my protein of interest tagged with mScarlet. After transforming my ligated product into DH5α bacteria and plating on LB agar, I noticed colonies with a pink tint to them. Miniprep pellets and even DNA elutions were also pink. The plasmid has been sequence verified and has the confirmed sequence. After looking online, I wanted to be sure this is not yeast contamination so the USER cloning was repeated. Again I saw both white and pink colonies and had pink pellets upon centrifugation. This issue has persisted across multiple batches of agar plates. Others in the lab have been doing transformations and have not come across this so it seems to be specific to my plasmid only. Could this be expression of mScarlet from my plasmid? This would be weird it is under the expression of mammalian promoter and none of my other mScarlet plasmids have had this issue. I've attached some photos for reference (arrows pointing to pink and white colonies).
don't panic!! this is not unusual: I got the same observation with sfGFP, redGFP...construct it depends on the plasmid construct probably in some of them there is a cryptic bacterial promoter and this fluorescent protein are so powerfull that even a small expression will give coloured pellet. more over
yeast will not grow at 37°C overnigt in LB and if you can get a plasmid from your culture it is most probably E. coli...
Based on the information and images you have provided, it is likely that the pink colonies and pink miniprep pellets are due to the expression of mScarlet, even though it is driven by a mammalian promoter.
If the ectopic expression due to stress were the primary cause, it would be unusual for the phenomenon to be consistently observed across multiple plates and transformations specifically for your plasmid while others in the lab do not experience the same issue.
Katie A S Burnette thanks for the helpful insight Katie! My only question is I have cloned several mScarlet plasmids (over 20 now!) and never once observed this. Just yesterday I transformed 2 mScarlet plasmids and none are pink so it seems to be something specific to this plasmid which is why it’s quite strange to me? And the fact the DNA elution was ever so slightly pink also concerns me…
Sara Milani Your assumptions are correct, The facts lead us in the same logic you have. Katie A S Burnette Maybe she did not really read your post, she just responded without thinking. It happens. The issue is almost assuredly more related to the specific plasmid or construct rather than just stress.
What sort of DNA extraction kit/method do you use? I'm assuming the pink is the mScarlet protein & it's not being washed away.
It might not matter if you are using the DNA for everyday protocol (e.g. restriction digest, PCR). Could always do a more stringent DNA extraction & cleaning protocol (old-style phenol/chloroform is pretty robust).
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