Dear all,
in a recent experiment on auto-induction, I included a control with only lactose as carbon-source for BL21(DE3). I expected the Colis to grow on this sugar, but the solution stayed clear... I repeated 3 times, all the same.
Is this particular strain unable to grow on lactose? This would not make sense, since in this case, auto-unduction would not work...
Can the vector I am transforming with in any way cause this result?
Can someone come up with an explanation for this unexpected behavior? ^^
Best regards,
Peter
Dear Peter, BL21 (DE3) has a mutation that blocks galactose utilization. The growth of the strain using lactose as the sole carbon and energy source is very poor. I recommend a period of growth in a medium with glucose or glycerol and then induce gene expression with IPTG. If you use the M9 must add an amino acid supplement.
To check lactose utilization ability of a strain, you can inoculate in MacConkey Agar. You can verify that the colonies of BL21(DE3) are observed as Lactose positive (red) between 16 and 20 hours. After this time the color change and negative colonies were observed. We recommend using the W3110 strain as a control.
When you provide the lactose as only source of carbon, bacteria gets very busy in producing the protein of interest and/or the T7 polymerase and don't care too much about the division. That is why in autoinduction media you have 5 sources of carbon: trypton, yeast extract, glycerol, glucose and lactose. So the cells are using glycerol and glucose first (which act as catabolic repressor for lac operon too) and only when those are depleted and biomass is there switch to lactose and ta da daa: induction starts. If you really would like to induce on sole lactose: grow the biomass in LB and then add lactose, in same fashion as you would add IPTG in classical induction
Julijus's answer is correct. If you have a good induction, bacterial growth stops since all the resources are devoted to expression, because that's how the system has been designed to work. You should first grow your bacterial in normal medium, without lactose, a small culture O/N, then in the morning expand to 40-100 times the volume and grow to OD 0.6-1.0 (also 1.2, depending on the medium you use, there are richer media that allow denser cultures, i.e. 2xYT, super broth, terrific broth), then exchange medium and use your lactose only medium: then a dense culture will start expressing and you'll get mgs of protein... well... in theory! with a bit of luck... ;-)
The construct should yield bovine Lactalbumin, so should not be toxic.
The cells should be able to just grow on lactose...
The cells grow normal on glucose, as well as on media containing glc/lac - but no induction is happening (no protein on gel).
Maybe the lactalbumin is toxic and selects for cells not expressing the gene?
Did you try plating untransformed cells / control into your new lactose plates?
Btw, what was the formulation for the media?
It's basically M9 medium - so Lactose is the only Carbon source.
The cells should be able to grow an Lactose... Since there is no induction of the construct (I checked) there is also no toxic product keeping them from thriving... Most puzzling o.O
Well, Peter, not necessarily... as Julijus wrote, until you have some glucose around, it will act as catabolic repressor so that even lactose import in the cell is blocked... which means no activation of expression.
So it probably depends on your experimental conditions the fact that you don't see expression... probably you look at a time point where you still have some glucose around (or it has been used up from too short time... to see proteins on a coomassie gel, you'll need an induction of at least 2-3 hrs).
Dear Peter, BL21 (DE3) has a mutation that blocks galactose utilization. The growth of the strain using lactose as the sole carbon and energy source is very poor. I recommend a period of growth in a medium with glucose or glycerol and then induce gene expression with IPTG. If you use the M9 must add an amino acid supplement.
To check lactose utilization ability of a strain, you can inoculate in MacConkey Agar. You can verify that the colonies of BL21(DE3) are observed as Lactose positive (red) between 16 and 20 hours. After this time the color change and negative colonies were observed. We recommend using the W3110 strain as a control.
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