[harrison]

Hi Matt,

1. I have never heard of a control board fail in that manner. Does it still happen if you disconnect the Beaglebone itself from the control PCB? I.e. just a bare, standard beaglebone sitting alone ont he desk?
If I were to guess the cause it might be that somehow the beaglebone’s internal power regulators got shorted, since some of these are used on the control PCB, but not sure how this could happen if as you say there was no liquid in contact.

2. Have you flashed the old beaglebone with the current version of the Chi.Bio operating system? This will update the underlying linux distro as well, which might solve this. I am unclear on what you mean w.r.t the specific board type: the Chi.Bio should be using a Beaglebone BLACK. Are you saying that you were shipped a Beaglebone GREEN? can you send me a picture of the board you _were_ sent?

The control board is designed for Beaglebone Black Rev C. I do not know whether it will work with other versions of Beaglebone Black, nor do I know whether it would work with beaglebone Green.
As to the pins – if they are going into the sockets as intended then it should be OK.

[harrison]

In principle – whatever you calibrate it for. Very high ODs will go beyond the _linear_ range of the normal setup, but you can fit a curve to compensate for this if you are mostly interested in that regime.

[harrison]

Let me know if any of that is unclear!

[harrison]

I hope my answer to your other post answers this: https://chi.bio/forums/topic/questions-regarding-od-measurement/#post-1251
If I recall correctly the confusion might be arising from inconsistent parameter names on my behalf, it looks like “CF” is the same as “P0” discussed in the paper (i.e. equation S3).

[harrison]

Dear Jeff,

P0 is meant to reflect the “raw power” of the excitation LED – i.e. to compensate for the fact that a LED in one reactor might be slightly brighter than that in another reactor.
M0 is how much scattering there is from the media itself – this is conceptually similar to the OD0 value from the Laser measurement approach (i.e. eqn s1 in the supplementary of the paper), which in that case reflects the absorption of the media.

You can see what these factors do by looking at Figure S10 middle or right column. In the top row you have the raw data – the transformation in eqn S3 is then used to get the second row. So, divide by approximately 1000 (ballpark value of P0), which takes our values of ~10000 to ~10. In the top row you see there is a non-zero baseline (i.e. the measured intensities are approaching some positive asymptote ~500 or there abouts) – this corresponds to M0. So, this is then removed in the second row and we see the asymptote is now the x axis (M0 ~0.5 in this case since we are subtracting it _after_ division by P0).

I think the approach I used was: To get P0 measure the amount of light when you just have a blank test tube in the reactor (and the appropriate LED is on) to give us an approximate raw power level of the LED. Then, put blank media into the reactor and measure this to give M0.

[harrison]

Australian mains supply power is compatible with the converters – so the easiest way would probably be to buy a UK-to-AU power adapter.

For example, like this

There should be stacks available online and from local electronics shops in Aus.

• This reply was modified 4 years, 9 months ago by harrison.

[harrison]

That’s a significant oversight! Contact Labmaker and let them know…

[harrison]

Thanks for the update François, that sounds about what I expect – given it is a single copy. One challenge of week-to-week reproducibility for small signals might be movement of the test-tube when you take it in or out. Your best bet might be to have it “auto calibrate” at the start of experiments (or whenever you expect there is ~zero fluorescence) or something like that.

Do your reactors have (white plastic) diffusers in their spectrometers? There could be a small white sheet just behind the apperture of the spectrometer (i.e. the circular part with hole in the middle). If not, I can mail you some – they can significantly improve signal-to-noise for small measurements (mostly it improves OD measurement, but also fluorescence to some extent).

Harrison

[harrison]

Hi Wijaya,

That is very strange. It is saying it is unable to connect to the thermometer on the control board (i.e. the one connected to the beaglebone).

Was your system working when it did have a pump board connected? If so then it seems the error must be somewhere in the code you have edited.

If not, the problem could be with thermometerExternal itself. That thermometer is the chip on the underside of the small “arm” jutting out of the control board, right next to where the ethernet plug is.
Can you take a look at it and see if it looks like it is soldered correctly (i.e. none of its pins are short-circuted to each other)?

[harrison]

Hi David,
Yes, that’s easy enough. Go into App.py and remove (or comment out) the lines in the turnEverythingOff function that refer to the pumps.

In particular, within that function comment out:
setPWM(M,’Pumps’,sysItems[‘All’],0,0)
SetOutputOn(M,’Pump1′,0)
SetOutputOn(M,’Pump2′,0)
SetOutputOn(M,’Pump3′,0)
SetOutputOn(M,’Pump4′,0)

As to using one pump module between two, yes, you can do this. Take a look at this thread:

Reprogramming P3 and P4 on the Chi.bio

I think Zoltan Tuza has nicely implemented this functionality here:
https://github.com/HarrisonSteel/ChiBio/pull/4/commits/fc5e468a894e59163fee93533b08a52d42c9ab22

That said, if you only have two devices and plan to stay that way, you could make a more “hacky” implementation that tells the setOutput function to activate Pump 3,4 on M0 whenever you command pumps 1,2 on M1 (i.e. assuming that the pump is connected to reactor M1).

[harrison]

Dear Diego,

Thank you for raising this. I’ve taken a look at trying to come up with additional fixes. Consequently, I have pushed a new version of app.py to the GitHub here:
https://github.com/HarrisonSteel/ChiBio/blob/master/app.py
Could you download this and try to run it on your device? It has several small changes that hopefully fix this issue (though it is difficult for me to diagnose it precisely).

That said, you are the first person I have seen have any new issue introduced after updating to the V2.0 software. Are these multiplexer-induced crashes only appearing after this upgrade?
Also, I am interested in what you said that “a restart of the beaglebone” is necessary to fix the problem. Does this mean that simply restarting the Chi.Bio software (i.e. with bash cb.sh) does not fix it? How about if you restart the chi.bio code (but leave the beaglebone on) and also power cycle the reactors? It seems very strange that there is a problem that requires the beaglebone to be power cycled, since this implies it may be somehow an issue with the underlying operating system (which should be pretty robust…). Out of interest what Control computer PCB version do you have (is it V1.0, V1.1, or V1.2?)

[harrison]

When you have been doing these past checks – I assume you have got the pump board connected to the reactor too, right?

Beyond that, I am not sure at this point – it seems as if there may be a more serious fault in the hardware somewhere. It could be in either the control board or the reactor – with only one of each it is difficult to diagnose which is not working. Most likely there may just be a poorly soldered connection that is corrupting the electrical signals. The best approach would probably be to contact Labmaker and ask if they can replace it, they are usually very good at this.
Sorry to not be of more help!

[harrison]

Hi John,

A few points/questions
– How “fluorescent” are the “appropriately fluorescent” cells? Do you mean they are at some level that (for example) is visible to the eye? How much expression did you measure from them as a fraction of e.g. “the brightest GFP E.Coli you have ever seen”
– You shouldn’t need to manually switching on the excitation LEDs first. When you click “Measure FPs” it will turn them on itself (and then off again ater the measurement).
– For the GFP have you tried exciting with the lower (395nm) LED?

Generally the system works best when you are not taking tubes in and out of the reactors. There is some wiggle room around the tube and this can impact absolute values of readings. Furthermore, when you start/stop stirring and heat/cool the reactor these will all make some (small) impact on the measurement. Instead, what the system is really designed to do is to track a single test tube (and its contained culture) over time. This is what is done in Figure 5 of the PLoS Biology Paper! So, to best assess your setup I would either a) Test with an inducible construct, which you can maintain at a set OD and then induce and observe fluorescence production or b) Grow your GFP cells to a fixed OD and maintain them there for some time, then pipette into the reactor concentrated RFP cells (say, with the goal of the resultant culture being 50% GFP and 50% RFP) and see how this changes the GFP/RFP balance measured over time.

[harrison]

OK. I must say I still think that liquid problem is the most likely. When you look at the top of the reactor you can see there are two sets of silver tracks closely spaced. Some around the middle hole where the test tube goes, and others around the outside. Did you very vigorously wipe with alcohol both those tracks?
One alternative would be to take two of the sides (i.e. the left/right, not the sides that the cables connect to) and then unplug the top sensor track internally (it should be obvious how to do this given the cabling) and see if that fixes it.

[harrison]

Hi Tatsuya,
You said it worked well, but then at some point it started showing this error – is that correct?
Does this error come if one specific reactor is connected, or is it happening when any of your reactors are connected?

If it is only happening with one specific reactor I think the most likely cause could be that there is some liquid that has dried on the top part of the reactor, where there is a circuit that is used to sense moisture overflow. If you have any liquid on this (or if it spilled and dried partially) it can trigger the circuit. To fix this I would suggest using a cloth with some alcohol and carefully wiping down the silver tracks on the top of the devices to make sure nothing is there that could be triggering it.

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