플라스미드 상의 lacIq 제거하기: SHuffle K-12와 Lambda Red Recombination

 

Q: 실험 조건 상 균주의 genome에서 lacI를 deletion하려고 하는데, SHuffle T7 Competent E. coli의 경우 플라스미드 상에 lacIq가 포함되어 있음을 확인하여 문의드립니다. 플라스미드에 관련된 정보는 구매처 상에서 더 확인할 수 없어서, 혹시 시퀀스나 해당 플라스미드를 curing할 수 있는 관련 정보를 전달해주실 수 있는지 문의드립니다.



A: SHuffle T7 Competent E. coli 버전 (NEB#C3026) 은 F' episome을 가지고 있으며, 여기에는 lac과 lacIQ가 포함되어 있습니다. 이 세포들은 lac 때문에 Xgal 배지에서 파란색으로 나타납니다. 만약 충분히 많은 양의 세포를 도말하면, F'를 잃어버린 극히 드문 세포들이 흰색으로 나타날 수 있습니다.

그러나, 두 종류의 SHuffle 세포 모두 세포질 DsbC (cDsbC) 를 발현하도록 설계되었으며, 이는 cDsbC와 lacIq을 포함하는 플라스미드를 통합하여 발현됩니다. 람다 레드 재조합 방법 (the lambda red recombination method)을 사용하여 염색체상의 lacIq을 제거할 수 있습니다.
 
SHuffle 게놈의 서열은 다음 링크에서 확인할 수 있습니다. 
Complete Genome Sequence of the Engineered Escherichia coli SHuffle Strains and Their Wild-Type Parents - PMC (nih.gov)
(Nucleotide sequence accession numbers 내용을 확인하시면 됩니다.)
 https://www.neb.com/en-us/faqs/2016/04/25/is-the-genome-of-shuffle-strains-available


#lacI 제거, #SHuffle K-12, #플라스미드 curing, #Lambda Red Recombination, #cDsbC 발현



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