SoluBL21(DE3) host strain

CSB investigators interested in testing this E. coli host strain can obtain free aliquots by contacting Heather Darling. Please read all the notes below as these cells do not behave like regular BL21s.


  • Evolved variant of E. coli BL21(DE3) that contains 'uncharacterized mutations obtained through special selection criteria.'
  • Improves expression of target proteins in soluble form.
  • Improves expression of toxic clones that cannot be established in BL21(DE3) or BL21(DE3)pLysS.
  • See Genlantis web page for complete details.
  • Transformation efficiency (3/9/11) = 3.6 x 107 cfu/ug
  • SoluBL21(DE3) variant made by CSB.
  • Contains the pRARE2 plasmid (isolated from EMD4Biosciences Rosetta 2 host strain) which supplies tRNAs for 7 rare codons: AUA, AGG, AGA, CUA, CCC, GGA, and CGG.
  • Chloramphenicol resistant.
  • Transformation efficiency (3/24/11) = 4.0 x 107 cfu/ug
The Genlantis transformation and expression protocol can be downloaded here as a pdf file.

Our notes on the transformation protocol:

  • The CSB cell aliquots are 100uL (not 50uL as sold by Genlantis). This is enough for 2 transformations.
  • The cells seem heavier than normal BL21s--they tend to sink to the bottom of the tube, so if you are splitting them into 2 tubes, be sure to mix them well before aliquoting.
  • As stated in the Genlantis protocol, you should plate all of the cells from a 50uL transformation, otherwise you may not get enough colonies.
  • Altering the heat shock time did not result in higher numbers of transformants.
  • We modified the Genlantis transformation protocol slightly:
    • Add 1uL of miniprepped DNA to 50uL of thawed cells.
    • Flick tube to mix.
    • Incubate on ice for 15 min.
    • Heat shock at 42°C for 45 sec.
    • Place on ice for 2 min.
    • Add 0.25 mL SOC medium. Incubate at 37°C for 1hr with shaking.
    • Microfuge cells at low speed (3000 rpm) for 3 min to pellet the cells.
    • Remove ~200uL of supernatant.
    • Gently resuspend cells in the remaining supernatant and plate the entire contents on LB agar plate containing appropriate antibiotic(s).
Our notes on the expression protocol:
  • We found that the Genlantis protocol for expression (M9 medium, room temperature) works, but it takes days for a starter culture to reach visible optical densities. (The staff at the Berkeley Macrolab facility also noted extremely slow cell growth and small cell pellets when using the Genlantis protocol.)
  • The Harrison lab at Harvard has had good luck using the SoluBL21(DE3) just as you would regular BL21(DE3) cells (e.g. inoculate 1L culture with 5-10ml of starter culture, grow at 37°C for 3-4 hours until OD(600nm)=0.6-0.7, shift to 18-20°C, induce with IPTG and express overnight. They found that when the SoluBL21 cells made a difference, it has worked in LB, TB or 2xYT and that the cell pellets are of normal/large size.
  • We have verified expression of a control protein using our SoluBL21(DE3) and SoluBL21(DE3)pRARE2 cells in both rich (2xYT) and minimal (M9) media induced with IPTG.
  • We have not been able to see any expression product when using SoluBL21(DE3) or SoluBL21(DE3)pRARE2 cells in Studier autoinduction media (either rich or minimal). We're not sure why this is the case but the result is reproducible, and we ran appropriate controls to ensure that the problem was not due to the media, plasmid, other reagents, our comp. cell prep. or harvesting too early.
  • Although we have not done extensive testing, in general we have not noticed a marked improvement in expression using SoluBL21s. However, in one case it did cause the protein to switch from insoluble to soluble expression.
Other observations/comments:
  • If you inoculate cultures with a clump of cells (instead of single colonies), the cells do not disperse even with vigorous shaking. You just end up with clumps of cells that sink to the bottom of the tube. It's weird.
  • I've noticed cell settling even when taking OD readings.
  • We were unable to see expression of a control protein in pLysS and pRARE2LysS variants that we made, so we are not releasing them.