[harrison]

Hello,

It is possible to use Chi.Bio as fed batch, but the challenge (as you have identified) is getting liquid addition/removal to be highly accurate. We used low cost pumps in the system to reduce price, so they cannot accurately deliver small volumes. One thing you could do is use some different pump that runs separate to the core platform, and just set it up to remove liquid from the reactor (e.g. with a thin needle through the lid) over time.
As to automated sampling, there are some commercial approaches to this and also DIY ones. See for example this toolbox which works with Chi.Bio
https://www.nature.com/articles/s41467-022-31033-9
Or this approach which uses a liquid handling robot and could be similarly adapted to the Chi.Bio reactors for precise and automated sampling:
https://www.biorxiv.org/content/10.1101/2022.06.13.495893v1

Harrison

[harrison]

Yes – if you leave them constitutively expressing it for a long time they can produce a lot per cell, more than the equilibrium amount when they are in exponential growth. But if you dilute them a lot at the beginning much of this shoudl be diluted out.

I would say in general measurements at extremes are difficult, either zero OD (where light is just passing right through, so you are just measuring the leakiness of the excitation light into the emission filter) or very high OD (where light being both scattered and blocked). If you want to get a good idea for what you are seeing the best bet is to grow non fluorescent cells at fixed OD for a while, then either induce them (chemical inducer or whatever your system is), or dilute them with fluorescent cells. That way you will have a very clear cut difference between off and on, and be able to accurately measure the dynamic range of the system for your biology.

[harrison]

What is happening to the culture BEFORE the experiment? Are the cells at stationary or exponential phase? If you are sampling from stationary phase then it can be that the GFP/cell at that point is already at or near maximum, hence the fluorescence/cell (which the emission/baseband measurement attempts to approximate) may already be at a very high level.

As to the long term (high OD) behaviour, it is difficult to conclude too much since when you get to high ODs there are multiple conflicting factors that determine the measurement. In particular, your excitation light (e.g. baseband) may scatter multiple times or be absorbed before leaving the test tube (i.e. to be seen by the sensor) and hence you get this decrease.

That said, most of these problems go away if you have the culture at a fixed OD – is this eventually your goal, or are you primarily interested in looking at growth curves from stationary-exponential-stationary phase?

[harrison]

It does look like a hardware fault to me, perhaps in the circuits on the PCB on the side of reactor that controls the temperature regulating heat-plate. If this was a bad connection then one would expect something like what you see. So, it is likely not the cabling at fault.
I’d have to say a likely best bet is to contact labmaker and tell them the hardware may have a manufacturing defect, and link them to this thread so they can see the evidence.

[harrison]

Hello,

Yes, by adding that dash I can now see it.
I think, contrasting your previous post, to me it looks like the EXACT same thing is happening in both the low and medium inductions for early times (i.e. until you start regulating OD). They behave basically the same (i.e. initial decrease), but the medium one is at least induced enough so that you can see it rise (whereas the low one is not enough for this) once they reach fixed OD.

Another possible explanation could be other non-linearities in the normalisation. For example that light scattering properties at the two wavelengths change as the OD changes, hence it appears to go down (slightly) over time. In general, the system won’t be great if measuring either very low fluorescence levels per cells, or measuring fluorescence at very low cell densities, since in both cases the bleed-through of the excitation to emission filters is drowning out the signal.

Generally our team does not find this a problem since usually what we do is grow cells to a fixed OD and hold them there for at least 5-10 generations (so that any dependence on previous culturing or state is mitigated), and THEN add inducer chemicals (or light, whatever). Is there a reason why you are not doing it this way? Trying to separate out the behaviour of genetic circuits from the impact of changing cell density is always challenging – hence why we make turbidostats in the first place.

[harrison]

One thing I often observe is a fluorescence DECREASE at the start of experiments if inoculating with cells that have come for stationary phase, or even from some other condition where fluorescence levels might be high. Even for promoters that are theoretically uninduced, often at stationary phase there is a lot of leakage, and if they haven’t been diluted this gives lots of time for them to accumulate fluorescence. Then, when you put them into a reactor (or plate reader) to grow you see fluorescence decrease initially as this previously built up level is diluted. This is less obvious if you are doing high levels of induction since the (desired) production is greater compared to the leak production.
DO you think this might explain what you are observing? Potentially I misinterpreted your description…

[harrison]

Hello Monika

When you start the software with the one that works, does it say that it recognises a reactor at port M0 (or whatever port it is?). This message would appear before the “Start Up Complete” message.

The second problem you mentioned – indeed if you have it plugged into a different slot you need to FIRST click on the active slot of the eight in the top left, THEN run commands. But, this would only be important if it is working.

Which brings me to the most important point; it seems in general as if there may be some significant hardware issues there preventing the system from operating. That is, defects in the manufacturing. It would be worth trying a comprehensive check of all the cabling connections – for example try different microUSB cables for each connection, different power supplies for each reactor, etc. But, if you still can’t get it to start I think you need to notify Labmaker and ask them to fix or replace the reactors and potentially even the control computer (it is difficult to identify WHICH is defective if it can’t start at all!).

I am sorry that this issue is causing a headache!

[harrison]

This is a tricky one, Mark. We sized the reactors to fit exactly with the ones on fisher, since one needs a nice close fit so there is little possibility of mis-alignment.
If you buy small quantities of the (theoretically) the same vials from other locally available manufactuers you can probably find something that fits. If all else fails we could ship you 20 or something Fisher ones.

[harrison]

Hello John,

It might be a possibility that the moisture sensor is causing some such problems. Have you ever seen condensation forming on the vial? It might happen depending on what the climate/humidity is like in your lab…

But, I’d have to say more likely is it is an issue with the underlying operating system which is fundamental to the Beaglebone. I have had one device in the past that gave similar PREEMPT SMP ARM error and it was fixed by flashing the beaglebone with a clean operating system, i.e. following the software setup instructions on this site. I don’t know if some beaglebones (the microcontroller) are themselves less reliable than others, but my experience indicates that might be the place (some cause issues for no apparent reason, potentially pointing to manufacturing defects). So, if none of the above works you could also try changing the beaglebone for which you can find a spare fairly cheap at many places online…

Harrison

[harrison]

Hello – I heard back from some US users on this point. They said they had purchased from Altec directly, or alternatively, that you could find some very similar size tubing (in imperial units) from usplastics.com. Let us know if you figure out anything else on that front!

[harrison]

Have you tried different stir bar designs as I mentioned above? You can get variety packs of different stir bars (e.g. of eBay) and then pick one that gets along with your reactor.

[harrison]

Alas I think that means it doesn’t have the fix. You could take the sides off and look if you desire – the change we are looking for is whether there is a 3d printed part approx 1cm high holding the magnets in the bottom above the fan.

[harrison]

Hello, I am not sure exactly which reactors do/don’t have it rectified since they are put together at the manufacturer so I don’t see the interior. Could you tell me the height of the reactor – measuring just the PCB/body of it (i.e. ignore the screws/feet on bottom) so I can investigate?

Regardless, the best option would be to follow the advice above, and attempt different stir bars.

[harrison]

Hello,

– In your graphs, I agree with you at some point it seems that the OD regulation algorithm decided it was going to stop running. But, it seems the input pump rate didn’t spike, i.e. the system didn’t THINK it ran out of media or got contaminated or such. This indeed seems like the OD regulation code simply stopped running – but it is very unclear to me how this could happen whilst the system keeps running as a whole.
I suppose you or anybody else didn’t press any buttons around this time that might have changed things? When you checked in the morning did the OD algorithm SAY it was running (ie.. the regulate OD button is blue)? If that was selected/blue then you shouldn’t be able to control the pumps manually since the OD Regulation algorithm blocks the user from changing pump rates (since they are being used to regulate OD).

– The top height of liquid in your fresh media and waste should be BELOW the top height of liquid in your reactor. Can you tell me where you read that it should be above? Is there some part of the manual that is ambiguous that I might fix? The reason they should be BELOW the reactor height is so that if a pump breaks the liquid won’t flow into the reactor. To achieve this in your current setup you need to put your reactor on a small raised surface (e.g. a used tip box or something) so it is higher than the media.

Harrison

[harrison]

Hello Shalni,
It should be almost impossible for the automated control algorithm to overflow the reactor, even if the OD readings are wrong, unless the tubing or pump is malfunctioning.
In particular, have you made sure that the liquid level in the reactor is always significantly above that in the fresh media and waste media containers? Furthermore, have you tested the pumps to check they make a good seal, and if not, added tape to the pump heads as shown in the troubleshooting guide?

• This reply was modified 3 years, 11 months ago by harrison.

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