I have recently been conducting experiments with the expression of several fluorescent RNA aptamers using E. coli (BL21). However, when I induced the expression using IPTG, the bacterial solution did not form a homogeneous suspension after induction. Instead, the bacteria aggregated into clumps, and fluorescence imaging of the aptamer under these conditions yielded very poor results. I attempted to avoid this situation by modifying the induction conditions, such as reducing the induction time from 4 hours to 2.5-3 hours, using 2 ml tubes for induction, and selecting individual colonies for post-culture induction. Unfortunately, all of these attempts failed to prevent bacterial clumping.
The induction experiment conditions were as follows:
Culture medium: LB medium
Strain: E. coli BL21 (DE3)
Induction conditions: 1 mM IPTG, incubation at 37 degrees C for 2-4 hours.
Does anyone know the reason behind this? Thanks！！！
Are the bacteria still alive after induction? If you plate them, do they grow?
I concur with Adam B Shapiro that you need to assess if they are still alive. That looks to me as if the cells are lysing, not aggregating. But it is hard to tell from a single picture
Thank you Adam B Shapiro Michael J. Benedik very much for your suggestion, I will apply the bacterial solution to the plates to verify that they are still alive and re-transform the new BL21 to repeat the experiment. Additionally, in order to give you a better understangding issue I mentioned,I have added the bright-field images taken under the Olympus confocal microscope (100x objective), and the inducing experiments below:
I transformed the pet28a plasmid expressing fluorescent RNA aptamers into BL21 bacteria, cultured them overnight on a LB agar plate (kanamycin), then selected a single colony and shaken it in LB medium for 12 hours, diluted it (OD600 < 0.4) and induced it in LB medium for 3-4 hours, and after inducing, I found the "bacterial aggregation" as shown above.
Image "Negative control": Only the induced bacteria were diluted and added to M9 medium without adding any additional reagents except kanamycin.
Image "Methylthioadenosine Phosphorylase Inhibitor added": Methionine was added during inducing and Methylthioadenosine Phosphorylase Inhibitor was added 3 hours before imaging.
Image "Normal picture": As a positive control, normal bacteria should look like this after induction.
Once again, I appreciate your valuable input.
Note that there will certainly be some cells that are alive in your culture, but you should make serial dilutions to be sure that most of them are alive. Be sure you are somewhat quantitative here.
If your two images with cells (not the negative control) are from similar density cultures, then there is a vast difference in number of cells in the two images. But the cells in the aggregated culture do look elongated relative to the control.
It sounds like you can measure fluorescence as an assay of expression? If so, why not take measurements over time after addition of IPTG. Obviously do a before induction control, then maybe 5 min, 10min, 20 min 40 min and 60 min and see what is happening over time.
This could be a bacterial biofilm, are you sure it is a pure culture and kanamycine resistant, did you subculture this from plate contained LB agar & kanamycine if so then you need to check toxicity of the induction component to this bacterium by subculturing it in LB with them.
Hope this helps
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