Some technical notes

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Ross, David J. (Fed)

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Dec 9, 2016, 11:05:32 AM12/9/16
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I got my first day in with the Attune flow cytometer yesterday. Did some practice runs with the E. coli strains Nick sent.

 

Some initial thoughts on measurement assurance steps that I would recommend adding to any protocols:

 

1.       Run buffer, focusing fluid, and media blanks. M9 media, in particular has the potential to form crystals that are similar in size/scattering to E. coli.

I would actually recommend running a media blank through the entire growth, dilution, and sample preparation process. So for the ‘Nielsen’ protocol we’re running: add one or more M9 media blanks to the plate for the day-0 overnight culture; then carry those media blanks through the entire process as if they were real samples, and measure them on the flow cytometer to check for issues with sample cross-contamination and/or other particulate. Also, before the 20x dilution into PBS, run a PBS blank on the flow cytometer, as a quality control check.

2.       Measure the sample carryover. As part of the protocol development for each instrument, after each E. coli sample, run a focusing fluid blank sample to check for carryover. Determine what rinsing protocol between samples is adequate.

Ariel- with the Attune instrument, running tube samples, it looks like running ‘Sanitize SIP’ 2 times between samples is a decent starting point for eliminating carryover.

John Sexton

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Dec 9, 2016, 3:39:49 PM12/9/16
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I recently consolidated the existing protocol documentation into the protocol_ecoli Google doc in the `2016_mini_interlab\protocols` folder, and I would encourage any sanctioned protocol modifications to make their way into that document in some form to avoid chasing them around in email.

David, these seem like reasonable assurance steps to me.

-John

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Jake Beal

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Dec 13, 2016, 5:19:26 AM12/13/16
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Thank you, John!  I second the strong desire to maintain all current protocol information in a single location.

I will repeat one of my computer science maxims here: "If it's not in the repository, it doesn't exist!"

Thanks,
-Jake


On Fri, Dec 9, 2016 at 8:39 PM, John Sexton <john.t...@rice.edu> wrote:
I recently consolidated the existing protocol documentation into the protocol_ecoli Google doc in the `2016_mini_interlab\protocols` folder, and I would encourage any sanctioned protocol modifications to make their way into that document in some form to avoid chasing them around in email.

David, these seem like reasonable assurance steps to me.

-John


On Fri, Dec 9, 2016 at 10:05 AM Ross, David J. (Fed) <david...@nist.gov> wrote:

I got my first day in with the Attune flow cytometer yesterday. Did some practice runs with the E. coli strains Nick sent.

 

Some initial thoughts on measurement assurance steps that I would recommend adding to any protocols:

 

1.       Run buffer, focusing fluid, and media blanks. M9 media, in particular has the potential to form crystals that are similar in size/scattering to E. coli.

I would actually recommend running a media blank through the entire growth, dilution, and sample preparation process. So for the ‘Nielsen’ protocol we’re running: add one or more M9 media blanks to the plate for the day-0 overnight culture; then carry those media blanks through the entire process as if they were real samples, and measure them on the flow cytometer to check for issues with sample cross-contamination and/or other particulate. Also, before the 20x dilution into PBS, run a PBS blank on the flow cytometer, as a quality control check.

2.       Measure the sample carryover. As part of the protocol development for each instrument, after each E. coli sample, run a focusing fluid blank sample to check for carryover. Determine what rinsing protocol between samples is adequate.

Ariel- with the Attune instrument, running tube samples, it looks like running ‘Sanitize SIP’ 2 times between samples is a decent starting point for eliminating carryover.

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Ross, David J. (Fed)

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Dec 14, 2016, 6:07:21 AM12/14/16
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I put in my suggestions as ‘suggested protocol revision’ in the Google Doc. I didn’t overwrite the previous version of the culture protocol because I’m not sure how the group is handling getting a consensus for protocol changes.

 

From: sbsc-flow...@googlegroups.com [mailto:sbsc-flow...@googlegroups.com] On Behalf Of Jake Beal
Sent: Tuesday, December 13, 2016 5:19 AM
To: sbsc-flow...@googlegroups.com
Subject: Re: [sbsc-fc] Some technical notes

 

Thank you, John!  I second the strong desire to maintain all current protocol information in a single location.

 

I will repeat one of my computer science maxims here: "If it's not in the repository, it doesn't exist!"

 

Thanks,

-Jake

 

On Fri, Dec 9, 2016 at 8:39 PM, John Sexton <john.t...@rice.edu> wrote:

I recently consolidated the existing protocol documentation into the protocol_ecoli Google doc in the `2016_mini_interlab\protocols` folder, and I would encourage any sanctioned protocol modifications to make their way into that document in some form to avoid chasing them around in email.

 

David, these seem like reasonable assurance steps to me.

 

-John

 

 

On Fri, Dec 9, 2016 at 10:05 AM Ross, David J. (Fed) <david...@nist.gov> wrote:

I got my first day in with the Attune flow cytometer yesterday. Did some practice runs with the E. coli strains Nick sent.

 

Some initial thoughts on measurement assurance steps that I would recommend adding to any protocols:

 

1.       Run buffer, focusing fluid, and media blanks. M9 media, in particular has the potential to form crystals that are similar in size/scattering to E. coli.

I would actually recommend running a media blank through the entire growth, dilution, and sample preparation process. So for the ‘Nielsen’ protocol we’re running: add one or more M9 media blanks to the plate for the day-0 overnight culture; then carry those media blanks through the entire process as if they were real samples, and measure them on the flow cytometer to check for issues with sample cross-contamination and/or other particulate. Also, before the 20x dilution into PBS, run a PBS blank on the flow cytometer, as a quality control check.

2.       Measure the sample carryover. As part of the protocol development for each instrument, after each E. coli sample, run a focusing fluid blank sample to check for carryover. Determine what rinsing protocol between samples is adequate.

Ariel- with the Attune instrument, running tube samples, it looks like running ‘Sanitize SIP’ 2 times between samples is a decent starting point for eliminating carryover.

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Jake Beal

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Dec 14, 2016, 6:09:24 AM12/14/16
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Thank you, David; Nick, Brian: can you please review and merge?

Thanks,
-Jake


On Wed, Dec 14, 2016 at 11:07 AM, Ross, David J. (Fed) <david...@nist.gov> wrote:

I put in my suggestions as ‘suggested protocol revision’ in the Google Doc. I didn’t overwrite the previous version of the culture protocol because I’m not sure how the group is handling getting a consensus for protocol changes.

 

From: sbsc-flow-cytometry@googlegroups.com [mailto:sbsc-flow-cytometry@googlegroups.com] On Behalf Of Jake Beal
Sent: Tuesday, December 13, 2016 5:19 AM
To: sbsc-flow-cytometry@googlegroups.com
Subject: Re: [sbsc-fc] Some technical notes

 

Thank you, John!  I second the strong desire to maintain all current protocol information in a single location.

 

I will repeat one of my computer science maxims here: "If it's not in the repository, it doesn't exist!"

 

Thanks,

-Jake

 

On Fri, Dec 9, 2016 at 8:39 PM, John Sexton <john.t...@rice.edu> wrote:

I recently consolidated the existing protocol documentation into the protocol_ecoli Google doc in the `2016_mini_interlab\protocols` folder, and I would encourage any sanctioned protocol modifications to make their way into that document in some form to avoid chasing them around in email.

 

David, these seem like reasonable assurance steps to me.

 

-John

 

 

On Fri, Dec 9, 2016 at 10:05 AM Ross, David J. (Fed) <david...@nist.gov> wrote:

I got my first day in with the Attune flow cytometer yesterday. Did some practice runs with the E. coli strains Nick sent.

 

Some initial thoughts on measurement assurance steps that I would recommend adding to any protocols:

 

1.       Run buffer, focusing fluid, and media blanks. M9 media, in particular has the potential to form crystals that are similar in size/scattering to E. coli.

I would actually recommend running a media blank through the entire growth, dilution, and sample preparation process. So for the ‘Nielsen’ protocol we’re running: add one or more M9 media blanks to the plate for the day-0 overnight culture; then carry those media blanks through the entire process as if they were real samples, and measure them on the flow cytometer to check for issues with sample cross-contamination and/or other particulate. Also, before the 20x dilution into PBS, run a PBS blank on the flow cytometer, as a quality control check.

2.       Measure the sample carryover. As part of the protocol development for each instrument, after each E. coli sample, run a focusing fluid blank sample to check for carryover. Determine what rinsing protocol between samples is adequate.

Ariel- with the Attune instrument, running tube samples, it looks like running ‘Sanitize SIP’ 2 times between samples is a decent starting point for eliminating carryover.

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Ariel Haim Hecht

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Dec 16, 2016, 6:38:44 PM12/16/16
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Hi guys,

I’ve been looking through the growth protocol, and I have a couple of questions:

1) Day 1 looks like a really long day. It seems like a minimum of 11 hours (3+5+1+1 plus time in between steps). Realistically its probably closer to 12 with set-up/clean-up time. Is that really necessary? If we’re trying to develop a method that would be used by other people, this seems like a bit user-unfriendly. Why would people want to adopt this? I think it would help a lot if it were compressed to fit within a standard 8-hour working day. 

To that effect, do we need such an big back-dilution to start? And why the second dilution? We’re not inducing here. 

2) I’d propose modifying it to eliminate the second dilution. Grow for 5 hours from the first dilution, then transfer to kanamycin for an hour, then make the measurements. 

3) Why glycerol as the carbon source? Alec’s paper used glucose.

Thanks,
Ariel




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Ross, David J. (Fed)

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Dec 16, 2016, 7:03:50 PM12/16/16
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I’ll second that- We’re running our first data set today. Fortunately, Vanya was able to start things off this morning at 7am. Then she handed off to me this afternoon, but I’m just now finishing up. So, yeah, 12 hours.

Both of us mostly worked on other things for the part of the day we were here, so that made it ok for us, but it’s not really workable for one person to do without risk of extra errors.

 

Also, Ariel- once I get a chance to look at today’s data, I’ll probably have some recommendations for how to set up the plate experiment on the Attune (what we did today ended up being less than ideal).

 

From: sbsc-flow...@googlegroups.com [mailto:sbsc-flow...@googlegroups.com] On Behalf Of Ariel Haim Hecht
Sent: Friday, December 16, 2016 6:39 PM
To: sbsc-flow...@googlegroups.com
Subject: Re: [sbsc-fc] Some technical notes

 

Hi guys,

-John

 

 

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Ariel Haim Hecht

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Dec 16, 2016, 7:14:15 PM12/16/16
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Thanks Dave, looking forward to hearing what suggestions you have.

And you make a good point about the errors. It’s not just that 12 hours is very inconvenient. It also significantly increases the likelihood of errors.


I recently consolidated the existing protocol documentation into theprotocol_ecoli Google doc in the `2016_mini_interlab\protocols` folder, and I would encourage any sanctioned protocol modifications to make their way into that document in some form to avoid chasing them around in email.

Vanya Paralanov

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Dec 16, 2016, 9:44:56 PM12/16/16
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Thank you, Ariel. Dave and I have been having the same concern about the long protocol, since last year when we ran the RNA-FISH experiment using the Cello growth protocol...

Here are my comments in addition to your questions:

1) We ran it today with me and Dave staggering our times. I had a 12h day (as I ended up running another experiment during the incubation steps) and Dave was not yet done when I left... I agree with you that the large dilutions and long growth are necessary when induction is needed (also confirmed by Alec as the reason he did the protocol this way). We are not inducing in this interlab. According to Alec, it is important to measure the cells in early exponential phase, which his lab considers to be at OD600 = 0.02. In this interlab we have constitutive expression, so I think one dilution and growth to 0.02 should be OK. Considering that the strain and media are different, and the dilutions are different (Cello's second dilution is 678 fold, not 30 fold, and the PBS dilution factor is also different), I would like to understand better why the protocol for this interlab was set up the way it is currently. I am sure there are reasons behind it :-)

2) I think we could do 1 dilution and grow in less time to 0.02 OD. If not, why not?

3) I was wondering why glycerol too. 

Thanks,
Vanya

-John

 

 

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Alec Nielsen

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Dec 16, 2016, 10:35:06 PM12/16/16
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A couple of notes:

1) Using the growth protocol from the Cello paper, culture OD600 reaches 0.2. This is believed to be early enough that protein concentration is still at steady state.

2) I use glucose because the sensors I use are not catabolite repressed. Although AraC+PBAD is normally repressed in the presence of glucose, I use an AraC-C280* truncation mutant that reduces this effect substantially (in addition to IPTG cross-talk). That said, glycerol is a good carbon source for avoiding catabolite repression in many other sensors.

Alec

Vanya


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Ariel Haim Hecht

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Dec 16, 2016, 11:30:14 PM12/16/16
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Thanks for chiming in, Alec. A couple questions, for you or anyone else:

1) This is mostly for my curiosity—why do you do a second dilution before adding inducer? We typically do just one dilution, then add inducer two hours later. 

2) What happens when you measure at higher ODs? Surely the measurements would be different, I’m just curious what happens to the circuits then. And what’s the sensitivity of the measurement to OD600—how close to 0.2 do you need to be? 

-Ariel

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Alec Nielsen

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Dec 17, 2016, 12:29:34 AM12/17/16
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1) The rationale for diluting cells before inducer addition is simply so that cells are in exponential phase before manipulating the circuit state. It's a matter of controlling for cell growth in order to simplify understanding of circuit dynamics and gene expression.

2) As cells transition from exponential phase to stationary phase, the dilution rate due to cell division decreases and the concentration of molecules may begin to increase. While cells are growing exponentially, the balance of mRNA/protein production and degradation (which includes dilution) is relatively constant - this is what is meant by "steady state" and it's why response curves are measured in exponential phase. Beyond this window, response functions begin to shift as some molecules accumulate due to reduced cell division. If these accumulating molecules are transcription factors, the concentration of downstream regulated genes can change non-linearly as OD increases. 

The other timing constraint for layered circuits is that enough time must have elapsed for concentrations to have converged. 

Hopefully in the future, a combination of genetic control systems and cellular models that account for growth rate and cell size will allow for circuits that function across growth phases, environmental conditions, etc.

Alec


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Jake Beal

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Dec 17, 2016, 6:13:45 AM12/17/16
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A note of reminder about focus: this conversation is drifting far away from the measurement of fluorescence itself, and into the reproduction of biological culturing protocols. This test was supposed to only be a preliminary sanity check, and this explosion of protocol issues is an important part of why I have been encouraging a shift to shipping pre-cultured samples for comparison, so that the variation in our results are not dominated by the well-known difficulties in reproducing culturing protocols. 

Thanks,
-Jake


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