I'm performing FACS on e coli, but the cells are clumping together so each event is multiple cells.

I ran a control where I had one flask of e coli expressing GFP, and one flask expressing RFP. Run separately, I got a clean population of either high-GFP fluorescent events, or high RFP fluorescent events. If I mix 10uL of each population into 200uL TE buffer and immediately run that sample, all the events have high RFP AND high GFP fluorescence, indicating the cells are sticking together immediately upon mixing.

Cell line: ER2566 (NEB) F- λ- fhuA2 [lon] ompT lacZ::T7 gene 1 gal sulA11 Δ(mcrC-mrr)114::IS10 R(mcr-73::miniTn10-TetS)2 R(zgb-210::Tn10)(TetS) endA1 [dcm] Host strain for the expression of a target gene cloned in the pTYB vectors. Carry a chromosomal copy of the T7 RNA polymerase gene inserted into lacZ gene and thus under the control of the lac promoter. In the absence of IPTG induction expression of T7 RNA polymerase is suppressed by the binding of lac I repressor to the lac promoter. Deficient in both lon and ompT proteases.

Growth media: M9 minimal media

FACS running media: TE

I know there are anti-clumping agents for yeast and mammalian cells, but I haven't come across one for bacteria.

Can anyone recommend:

  • an e coli protein expression strain that is known to not clump

  • an anti-clumping agent for bacteria

  • any other possible solutions ?

Thank you!

  • $\begingroup$ Check out the (many) strains and methods in this paper: ncbi.nlm.nih.gov/pubmed/18926860 $\endgroup$ – Luigi Jan 21 '15 at 16:09
  • $\begingroup$ If you don't have access to that journal, I put a copy of the PDF in my Dropbox here. $\endgroup$ – MattDMo Jan 21 '15 at 16:38

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