recombination, why is it bad? - (Aug/29/2006 )

i electroporated my plasmid into BL21 strain of E-coli which seems to have recombinase. i have to do maxi-prep on them and my freind says it is not recommended since they contain recombinase. i understand recombination is something like crossing-over and stuff. how and why would it effect the plasmid?

-Kathy-

well in my Bl21 cells its recA1 so i know its a generan recombination deficient that ensures insert stability and helps prevent unwanted recombination between insert and host so thats why i know recombinase is bad.

-spanishflower-

thanx spanish flower, so i think my BL21 must be "origami" type that doesnt have this gene. so the actual recombination is between the insert and the host. thanx a lot for the clarification!

-Kathy-

not necessarily, although many vectors have sequences derived from e coli in the first place. so, the homology perhaps makes an easier place for recombination to occur? not sure though, does anyone know? how stringent is recA?

-aimikins-

QUOTE (Kathy @ Aug 29 2006, 11:06 PM)

i electroporated my plasmid into BL21 strain of E-coli which seems to have recombinase. i have to do maxi-prep on them and my freind says it is not recommended since they contain recombinase.

i understand recombination is something like crossing-over and stuff. how and why would it effect the plasmid?

if ur plasmid has some sequences like IRES or repeat sequences, these will recombine in recombinase positive strains and u could lose bits and pieces of ur vector and insert. some times u lose small fragments not noticable till its sequenced.

For such plasmids, we use strains which dont allow recombination.

-scolix-

thank you so much everyone! now im reading through these abbreviations of E-coli genome and wondering what is the difference between recA1 and F'?

-Kathy-

recA is a gene, F' ("F prime") is a conjugal plasmid. The F plasmids (for fertility) were one of the early discoveries about conjugation in bacteria; an F' plasmid is an F plasmid that has picked up a bit of the E. coli chromosome -- the discovery that F plasmids could transfer parts of the chromosome was hugely helpful in creating the first genetic linkage maps.

From Yale's History of Microbiology project:

QUOTE

The directionality and polarity of the bacterial mating process, first suggested by Hayes, greatly clarified the understanding of bacterial genetics as studied by mating experiments. The problem of sexual compatibility, however, remained. The rather rare property of a given E. coli strain to mate was a puzzle. In 1952 J. Lederberg, L.L. Cavalli, and E.M. Lederberg (Genetics 37:720-730) and in 1953 Hayes (J. Gen. Microbiol. 8:72-88) independently reported that the ability to act as a donor in a bacterial mating was a property controlled by an "factor" designated "F" (fertility) that seemed to behave as "an infectious particle."

[link]

A good source of genetic marker info is here.

-HomeBrew-

goodmorning kathy
can u do ur transformation in another host strain like DHalpha or c600 for example and then after purifiaction of ur plasmid use it in BL21( i guess u use this for protein expression) i do this transform to JM109 then purify by maxiprep and the transform my BL21 cell for my protein.

-spanishflower-

spenishflower, i used BL21 in protein expression same strain and had wonderful results even in minipreps. and here in the lab they didnt have any better choise i thought, but yesterday they discovered JM109 and so im redoing my work.. but speaking about protein expression, maybe you better try directly BL21, it worked wonders for me.

homebrew, thankx a lot for the links, so much more things make some sense now... have a great day all!

-Kathy-