It is possible that you are not able to get confluent growth/a bacterial lawn of recombinant E. coli TOP10 for a variety of reasons. Some of the potential causes could be that the cells have not been transformed with the desired plasmid, the cells are not competent, or the plasmid is not expressed in the strain. Additionally, if you are attempting to grow on an agar plate, the agar may be contaminated or not prepared correctly, or the plate may not be incubated at the right temperature or for the right amount of time. Lastly, the cells may be overgrown with a contaminant, or they may be over-expressed and unable to survive in the media.

At those cell densities you should easily be getting a full lawn. My first thought is that you have some contamination in your culture, perhaps phage for example. You might streak your stock strain out for clean single colonies for 2 cycles to be sure it is clean.

Secondly if you are using kanamycin plates for your lawn, maybe your strain is losing the plasmid when growing the culture so many cells are no longer kanamycin resistant. You could see if the lawn looks different on LB vs LB+kan.

Thirdly maybe something has changed in your procedure of how you are spreading. If you are using a hockey stick to spread maybe you are not letting it cool down enough. Or if you are using top agar then maybe it is too hot.

Bandla Ramesh I forgot to say that I started from a glycerol stock (stored at -80oC for 2 years) of my recombinant cells. I streaked for single colonies then inoculated liquid LB. Then I took aliquots at various OD600 readings and spread onto plates. I wasn't plating a transformation.

Michael J. Benedik I think your point about some of the cells losing the plasmid is the most likely cause. I will try your suggestion of LB vs LB+Kan. I also forgot to add that there is also 0.2% arabinose in my plates. This is because when I perform the disc diffusion test, I need the arabinose for induction of expression of the protein of interest (a suspected beta-lactamase). So, I figured I need to find what OD600nm, volume etc etc gives a lawn in the *same* conditions as I will be using in the disc diffusion test. Thanks.

Then you might also want to try a plate without arabinose to confirm that expression your gene is not the issue (although it shouldn't be in this case).

Michael J. Benedik update:

Yesterday I tried LB only vs LB + Kan vs LB + Kan + 0.2% arabinose.

The LB only is as one would expect.

The LB + Kan has a lawn (with a little gap of uneven coverage)

The LB + Kan + 0.2% arabinose has barely any colonies.

This was at an OD600 of around 0.5.

I'm now wondering if the arabinose % is too high and is causing the cells to overexpress at the expense of growth...?

I'm now going to try lowering the arabinose e.g. 0.02%, 0.002%

FYI I'm using a pBAD plasmid and 0.2% arabinose is what I've always used when doing protein expression and purification with this plasmid.

At least you have identified the problem. Normally 0.2% arabinose should be fine and pBAD does not normally express at an excessive level. But it does appear that your protein itself is slightly toxic, which is a surprise. It might be that this putative beta-lactamase is not being secreted efficiently (they are usually periplasmic enzymes) and the toxicity is coming from inhibiting the secretory pathway. This is a well known phenomenon.

You might just try plates with no arabinose and those 10 and 100 fold dilutions to see how they do. It does not require high level expression of beta lactamase to get resistance so you may observe resistance even without induction and you can do your disk tests under those conditions.

Michael J. Benedik Interesting...I got a lawn with 0.002% arabinose at 37 degrees (usual incubation temp) and 30 degrees.

I also was wondering if my spreading technique may be too vigorous (I use the sterile single use plastic ones but I spread very rigorously and it's probably a bit overkill).

I normally spread 50 or 100uL (when doing a transformation for example) but in these experiments I've even tried 1mL to see if that makes a difference. I will go with 200 or 250uL for these experiments once I can replicate the lawn with the lower arabinose conditions. Thanks again for your advice.

I doubt that you can be too vigorous. But 100-200 ul should be optimal, as long as the plates dry well.

good luck with the experiment.

Note that the low arabinose concentration you are using may not be very inducing, but worth trying the experiment