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Transformants/Revertants grow on plates but crash in liquid

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#1 foog



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Posted 04 October 2012 - 07:09 AM

I'm transforming Top10's with a ~9kb ligation. The vector is tolerated in E. coli, and the insert is tolerated in another vector. Transformation was performed with both heat shock and electroporation. IPTG induction of the insert in another vector produces expected results and significant protein production without any sign of the growth defect mentioned below. Non-insert ligations (all vectors were treated with O/N SAP) produce significant colonies compared to those with insert.

When plated on carbenecillin plates, potential transformants (and even revertants from the non-insert ligation) come up fine. Re-streaking produces colonies that appear stickier than the average streaks. Overnight liquid cultures in either amp and carb crash (lysates on the bottom of the tube), but there are survivors in the liquid as determined by OD and scoping. OD'd during the day over several hours to observe when they crash, and its nearly instant, with lysate pooling at the bottom as time progresses. Scoping shows E. coli with segregation defects (lots of 8mers rather than typically dividing 2mers). Swapped media and antibiotics to no avail and other constructs come up fine in the same media/antibiotics.


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