I constructed a plasmid containing a coding region with 2 mutation sites (genes for lysine and arginine were replaced to alanine) and transformed into Stellar E. coli cells. Following a 16h-incubation at 37°C, several colonies happened to grow, indicating the successful transformation. I further kept it in 4°C and found several colonies changed into pink-like color in the next day (see the attached pictures). I did the plasmid transformation using stellar cells a lot using wild-type plasmid and such color changes never happen. My questions would be:

1. Do anyone here experience it?

2. What is the possible reason for this?

3. Might it be due to the affect of mutations?

4. What are the reasons for color change of E. coli colonies?

Your comments would be very appreciated. Thank you!