[Nuha]

The decrease in fluorescence might make sense if we are inoculating cells at relatively high OD – closer to the target OD. But in my case I even started experiments directly from a colony on a plate and set the chibio to regulate it at OD 0.5, once it reaches that point. So there is no chance for cells to be at stationary – the cells are always in exponential phase.

I think it’s best if you view the 2 images I shared. I can see the ‘low induction’ link has been changed by the forum to 1 dash instead of 2 dashes between AD and 2, that is why it is not opening. Here it is in link format (I hope that works, else please just type it):

If you compare medium GFP induction with low GFP induction images I hope you’ll understand what I mean. I think the normalization is not sensitive enough at low fluorescence inductions and may potentially bias fluorescence measurements.

[Nuha]

The link doesn’t seem to be working for low induction. Here it is again:
https://drive.google.com/file/d/1VpzAD–2-R3vQupKj3H7jrQUFTmgvTE3/view?usp=sharing

[Nuha]

Apologies for getting back late to you, there was a delay in running the experiments and we recently received the latest chibios. I have switched to M9 media, thanks for the feedback.I’m also running the experiment starting at very low OD, and have the OD regulation set at a target OD 0.5; so I’m not diluting. Here is the interesting observation:

I noticed that for low GFP/RFP induction, the normalized fluorescence over time starts at a higher GFP value (OD 0.05). When the cells approach OD 0.4, GFP fluorescence starts dropping until it reaches the steady state as the OD is set at 0.5.

This behavior is not seen for medium and high GFP induction. Both medium and high normalized fluorescence values show the expected increase in slope from OD 0.05 to OD 0.5, and reaches steady state when the target OD is set at 0.5.

When I remove the normalization, low GFP induction is corrected and shows the expected graph like the medium and high GFP inductions. I have attached the graphs for your reference:

https://drive.google.com/file/d/1VpzAD–2-R3vQupKj3H7jrQUFTmgvTE3/view?usp=sharing

https://drive.google.com/file/d/1yTL252cIFNBfMEs7_12JxM5Qp2kXAhoG/view?usp=sharing

[Nuha]

I just replied but don’t seem to see it. I hope you received it?

[Nuha]

So it turned out there was downtime on the iMAC, when adjusted I stopped seeing the errors I mentioned thankfully. But, I do get one error not too often that stops the whole thing, “Failed to disconnect multiplexer”. This error also becomes more often when I connect more than 2 reactors to the beaglebone. I also get pump connection errors hours into the experiment sometimes, but rarely does it stop the experiment. Any suggestions to why that is happening? I’m thinking to try different beaglebones, and try a new computer afterwards to compare.

[Nuha]

I thought the same regarding the computer, but I disregarded it since (1) I’m using the same computer I used last year (which is an iMAC not PC) and it’s the first time I see the errors (2) if it has to do with inactivity I expect the disconnection to happen around the same time every time I run an experiment, but it is variable and it has only gotten worse (I ran an experiment before updating everything and it stopped after 26 hours, and after updating I’m getting these errors within an hour or less into the experiment).

Updates for the iMAC have also been installed recently, not sure if that plays a role. I’ll double check and try with a different computer just to be sure. Thank for the prompt response!

[Author]

Posts