[harrison]

The difference between that proposal and what I am suggesting is that my suggestion is pumping out/in at the same time, while keeping the volume constant.

In practice what you are suggesting (reduce volume then pump back in fresh media) will yield the same result as setting a specific OD reduction target (i.e. reduce OD by half) – is this not correct?

[harrison]

Hi Sina,
I must say the description confused me; would it be possible for you to write down precise steps you want to achieve point-by-point?

There is some ambiguity in what is meant by “volume reduction” in my mind. In particular, as the Chi.Bio is set up by default the “outlet” tube is only some way down into the reactor. It is set at a fixed height so that you can never reduce the volume in the reactor below the normal amount (20ml or so). That said, you can dilute it by adding fresh media and taking out a mixture of media + cells, but during that process the total volume remains (approximately) constant.

If you really want to reduce the volume this is possible (but would require changing the height of the tube and would not be very effective), but it is somewhat unclear to me what you would gain based on your description.

In my subsequent messages you referenced I suggested another approach which is to use the OD Regulation function already in the system. You could on a X hour loop do something like:
1. Measure current OD (say, it is equal to 1.0)
2. Turn on OD regulation with a target of 0.5 (i.e. half the measured value).
3. Wait until OD has reached 0.5 (probably will take 5 minutes, though pumps are only on a fraction of the time). At this point half the cells + media have been removed (and replaced with fresh media).
4. Turn off OD regulation and wait 12 hours before beginning step 1 again.

Does that make sense?

I am afraid I may have misinterpreted your specific goal, if so please do let me know what was meant 🙂

• This reply was modified 4 years, 10 months ago by harrison.

[harrison]

That is correct!
The software setup instructions in fact assume that you are starting with a completely stock-standard Beaglebone, so you should be able to follow them as written.

[harrison]

It does seem suspicious that it would stop working then magically start up later. My best guess would therefore revert back to the moisture sensors – perhaps the one half-way down inside the device was tripped.
Or, it might indeed be a hardware issue, perhaps temperature changes etc helped it re-make a good connection. Hopefully it keeps operating as expected…

[harrison]

Alternatively you could measure the power supply with a multimeter to check if it is as expected.
Probably this is an unlikely cause – given it worked previously, it seems more likely that the reactor/hardware might have broken (probably there is a bad solder connection) rather than the 12V packing in.

[harrison]

Hi Lachlan,

I’ll assume everythign is plugged in and powered on etc.
Maybe one approach would be to try a different 12V power supply, and different USB cables in case they are broken somehow.

Failing that I would have to say the most likely application is a fault in the hardware/assembly which is somehow preventing it from communicating with the reactor. I don’t think moisture is the issue (as you said) since usually if that is the case it wouldn’t reach the “start up complete” message without error.

Unless you want to get in the device and start soldering connections etc probably the best approach is to reach out to labmaker and ask if they can replace/fix it.

[harrison]

That’s a very good question. It probably is a foundational problem with the Beaglebone chip that it has half an operating system on it – potentially preventing it from booting.
I do not know how in particular to fix it, perhaps there are other beaglebone users (unrelated to Chi.Bio) who have had the same problem online. If it isn’t taking a new flasher SD card then perhaps your only option is to buy a new beaglebone (they are not particularly expensive on their own)!

[harrison]

If you run with my second suggestion (using turning on the RegulateOD function) then those things wont be an issue – it shouldn’t overflow or reduce volume.

We do not have a dramatically more documented version, if you want to learn more best bet is looking at the comments, description in the Supplementary Information of the paper about the platform in PLOS Biology, and the user guide. Making more documentation has been on my to do list, but it never reaches a very high position by the time other items come to overtake it…

[harrison]

Something else occured to me as a potentially strategy: If instead of turning the PUMPS on every 12 hours, you might do better to turn on the OD Regulation function even 12 hours, and have it set to (for example) try to reduce the OD to half of whatever it’s value is at that time. Then, have it do a check that turns off the regulation once that target is reached. This way, the pumps will always provide an appropriate amount of liquid (i.e. in terms of factor of dilution), which you would not achieve by just setting them to a fixed flow rate for a fixed amount of time.

[harrison]

No worries.
One practical problem you may encounter would be that the pumps will need calibrating to make sure they are injecting the same amount of media as each other. Without this I would guess it might vary by up to 50% between pump heads, since the pumps used are the cheapest ones available!

[harrison]

I am afraid if it is going to be that infrequent you will need to do it in the python.
Probably the easiest way to do so would be to add a conditional statement in the main loop of the code (the function runExperiment) which turns the pumps on every 12*60=720 cycles (since each cycle is nominally 60 seconds).
You could to this in a similar way to the function already there to code the terminal which looks like this:

if(sysData[M][‘Experiment’][‘cycles’]%10==9): #Dont want terminal getting unruly, so clear it each 10 rotations.
clearTerminal(M)

In your case you would want it to happen every 720 cycles (i.e. set the top line to have %720=1 or something) and then when that condition is true, set the pump rates to whatever you want and switch them on, and if the condition is false (i.e. add an else statement below it) turn the pumps off.

[harrison]

Hello,

When you click “start” it will probably automatically set the pump rate back to zero, and it won’t necessarily Switch it on.

If you want the pump to run MANUALLY then you need to set the rate to something non zero (like 1) and then click “Switch” on that pump to turn it on.

If you want it to be turned on/off automatically then you need to set it into Turbidostat mode (i.e. “Start” the experiment then click “Regulate OD”) and then provided that the measured OD is less than the OD set-point it will intermittently turn on the pumps.

Does this make sense?

[harrison]

Great, glad to hear it is all sorted!
Thanks for the pics – indeed it is often hard to tell whether there is a short – even when you are looking down the microscope yourself, but it dose seem like a major possibility given how much solder is on there.

Harrison

[harrison]

Hi Jesse,
Seems like you have been through quite an ordeal, but made very good progress!

You mentioned the TCA9803 was one issue – did the soldering look bad on the original one? I am suspicious of the assembly, more than the chip internals, as with your spectrometer issues.
It would be worth telling Labmaker that you had these exact issues, which likely arise from poor assembly, since they are ultimately an issue on their end and need to be looked at more broadly to reduce issues like this (for you and others) in the future.

As to what to do next – it seems you have most/all of it working now. To be honest, I have never established some kind of “stress test” procedure for the devices. I have ~20 in my lab which I soldered entirely by hand and have never had any strange issues with the electronics on any of them. Once they are running fine, at least in my experience, they stay fine (ours have been used intensely for almost 2 years continuously).

One way you might test the whole assembled thing (short of doing an experiment) is
1) put a test-tube with ~20 ml of water and a stir bar into the reactor
2) Calibrate OD such that whatever you have in there reads at ~0.5.
3) Turn on all the FP measurements
4) “Start” the automated experimental mode
5) Set the OD target to 0.2 (or anything <0.5) and activate the OD regulation - but leave the silicone pump tubes disconnected from everything.

Doing the above would make the device heat/measure/stir continuously as in normal operation, and would also run the pumps at high-ish speed (since they aren't connected to the liquid every minute they would attempt to pump air, and continuously do this since the OD target is less than what it is measuring - hope this makes sense).

[harrison]

I think that is reasonable plan and aught to achieve what you are aiming for!

Keep in mind that both devices (Chi.Bio and the spectrometer) may be inaccurate at high ODs since it enters the “multiple scattering” regime. For example, this is why generally you need to dilute samples before putting them into the spectrometer when at high ODs…

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