The BioBricks Foundation:Standards/Technical/Measurement/Promoter characterization experiment (FACS): Difference between revisions

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*5 mL polystyrene round bottom tube with cell strainer cap: BD Falcon #352235
*5 mL polystyrene round bottom tube with cell strainer cap: BD Falcon #352235
**'''[[User:Jason R. Kelly|Jason R. Kelly]] 13:37, 15 February 2008 (EST):'''What's the cell strainer cap for?  Don't normally use that.
**'''[[User:Jason R. Kelly|Jason R. Kelly]] 13:37, 15 February 2008 (EST):'''What's the cell strainer cap for?  Don't normally use that.
**'''[[User:CarolineAjo-Franklin|CAjoF]] 12:42, 18 February 2008 (EST)''': The cell strainer cap is make sure there are no big particulates in the sample that may clog the FACS. It is more important for fixed cells, but it certainly never hurts.
*[[Endy:M9_media/supplemented|Supplemented M9 Media]]
**'''[[User:Julius B. Lucks|Julius B. Lucks]] 18:49, 3 April 2008 (EDT)''': The exact ingredients we used for the minimal media can be found here: [[Julius_B._Lucks/M9_Supplemented_Minimal_Media]]


==Culturing of the strains==
==Culturing of the strains==
#Streak LB+Kan plates of the 5 test strains and an LB (no antibiotic) for TOP10
#Streak LB+Kan plates of the 5 test strains and an LB (no antibiotic) for TOP10
#For each test construct add 5ml of supplemented M9 medium (w/glycerol, [[Endy:M9 media/supplemented|see preparation protocol]]) and Kanamycin (20ug/ml) to X 17mm test tubes.  Add 5ml of supplemented M9 medium (w/glycerol) with no antibiotic for the TOP10 negative control cells to X 17mm test tubes.
#* '''[[User:Julius B. Lucks|Julius B. Lucks]] 19:02, 3 April 2008 (EDT)''': This was done for us by [[User:CarolineAjo-Franklin|CAjoF]]. Plates were stored in the 4C refrigerator for 2 weeks.
#For each test construct add 5ml of supplemented M9 medium (w/glycerol, [[Endy:M9 media/supplemented|see preparation protocol]]) and Kanamycin (20ug/ml) to three 17mm test tubes (so 15 tubes total).  Add 5ml of supplemented M9 medium (w/glycerol) with no antibiotic for the TOP10 negative control cells to three 17mm test tubes.
#*how many replicates do we want to do (X)?
#*how many replicates do we want to do (X)?
#*I would suggest 3 to start. If the replicates are wildly different, we can move to more.'''[[User:CarolineAjo-Franklin|CAjoF]] 12:45, 18 February 2008 (EST)'''
#Inoculate media by picking single colonies from plates.  Grow cultures for 20 hrs at 37°C with spinning at 70 rpm.
#Inoculate media by picking single colonies from plates.  Grow cultures for 20 hrs at 37°C with spinning at 70 rpm.
#*'''[[User:KimdeMora| Kim de Mora]]''' I had trouble using culture sticks to innoulate the first time I did the experiment.  There was a lot of debris that was picked up by the machine.  Works much better with tips.
#*'''[[User:Dileep D. Monie|Dileep D. Monie]] 13:49, 1 April 2008 (EDT)''': 70 RPM seems a bit slow. Any reason why I shouldn't shake more vigorously?
#**'''[[User:Jason R. Kelly|Jason R. Kelly]] 14:47, 1 April 2008 (EDT)''': I think you should be OK using whatever RPM you normally use.  The variability between shakers/rollers makes me guess we couldn't easily standardize this if we wanted to.
#In the morning dilute 100x into 5ml of '''pre-warmed (37C)''' fresh media to get cells back into log phase and let grow for 4hrs under same conditions as overnight
#*Does the back-dilution use media containing antibiotic for the 5 test strains? '''[[User:CarolineAjo-Franklin|CAjoF]] 16:11, 29 February 2008 (EST)'''
#**'''[[User:Jason R. Kelly|Jason R. Kelly]]''':Yeah, they should contain Kan.
#The cultures should be at an OD of 0.2 - 0.4 after 4hrs.


1. Grow an overnight culture (20hrs) in 5ml tubes on roller in
==Live Cell FACS Procedure==
supplemented M9 @ 37C
#When your 5 mL culture reaches OD600 ~ 0.300, pellet the cells by centrifuging 1ml of each culture in an eppendorf at 3000xg at room temperature for 5 minutes.
2. In the morning dilute 100x into 5ml to get cells back into Log
#*'''[[User:KimdeMora|Kim de Mora]]''':  I did this with an OD of 0.1 and with 250 μL of washed cells, still had 90 000 events/sec on our LSR-II.-
phase let grow for 4hrs
#Remove the supernatant.
3. Take OD600 and then dilute cultures to OD 0.07 in 5ml media
#Resuspend the cell pellet in 1 mL PBS.
(pre-warmed to 37C)
#Add 250 uL washed cells through the cell strainer lid into a 5 mL polystyrene tube. To get the solution to pass through the strainer, apply slight pressure to the strainer lid with the pipet tip as dispensing the cell solution.
4. Let grow for 1hr should be at an OD of 0.1 - 0.15, take cells and
#Add 250 uL PBS through the cell strainer lid into the 5 mL polystyrene tube.
put immediately on ice
#Place cells on ice.
5. spin down and resuspend in PBS and then you can run on flow
#Analyze cells by flow cytometry using 488 nm excitation as quickly as possible.


 
==Analysis of FACS Data==
 
#Using the signal from the TOP10 cells, create a gate (P1, in image below) in the forward scatter-side scatter plot that captures the core of the scattering events.
# After 20 hrs, cultures were diluted 1:100 into 5 ml of pre-warmed fresh media and grown for approximately four hours under the same conditions(17mm tubes, warm room, spinning at 70 rpm).
#Using the signal from the TOP10 cells, create a second gate (P3, in image below) on the fluorescence channel which includes 99% of the total population and a third gate (P4, in images below) which encompasses all greater fluorescence signals.
# After four hours, a 500ul aliquot from each culture was then used for OD600 measurement.  The cultures were diluted to OD 0.07 in 5 ml of pre-warmed fresh media to be grown for one hour under the same conditions(17mm tubes, warm room, spinning at 70 rpm). 
#Copy these gates to all other samples.
#For each culture a 200 µl aliquot was transferred into a flat-bottomed 96 well plate (Cellstar Uclear bottom, Greiner).  An additional control of blank media was also added in the same aliquot.  Three replicates (three aliquots of 200 ul) of each culture were measured.  
#Determine the geometric mean of fluorescence intensity of the P4-gated population in each sample, <I<sub>sample,P4</sub>>.
#The plate was incubated in a Wallac Victor3 multi-well fluorimeter (Perkin Elmer) at 37°C and assayed with an automatically repeating protocol of absorbance measurements (600 nm absorbance filter, 0.1 second counting time through 5 mm of fluid), fluorescence measurements (488 nm excitation filter, 525 nm emission filter, 0.5 seconds, CW lamp energy 12902 units), and shaking (1 mm, linear, normal speed, 5 seconds).  Time between repeated measurements for each well was 2 min and 21 s.
#*'''[[User:Dileep D. Monie|Dileep D. Monie]] 18:38, 3 April 2008 (EDT)''':The geometric mean of I20259 should be calculated using the P3-gated population.
#Data processing was used to calculate the GFP synthesis rates for each well. The data for each construct tested was averaged across the three replicates wells and the three replicate cultures.
#Determine the geometric mean of the P3-gated population of the TOP10 cells, <I<sub>TOP10,P3</sub>>. This corresponds to the mean background fluorescence intensity of the cells.
 
#Calculate the fluorescence intensity in consensus Biobrick GFP units = I/I<sub>I20260</sub> = <I<sub>sample,P4</sub>>-<I<sub>TOP10,P3</sub>>/(<I<sub>I20260,P4</sub>>-<I<sub>TOP10,P3</sub>>).
 
[[Image:Mfa091001_forwardSide.jpg|500px|thumb|left|side scatter vs. forward scatter dot plot for TOP10 cells, showing gated populations corresponding to cells]]
 
[[Image:Mfa091001_FITC.jpg|500px|thumb|center|Histogram of raw GFP fluorescence/cell for TOP10 cells, showing gates created with this negative control]]
==Live Cell Procedure==
[[Image:Mfa091010_FITC.jpg|500px|thumb|right|Histogram of raw GFP fluorescence/cell for I20260 cells, showing copied gates.]]
*When your 5 mL culture reaches OD600 = 0.100, pellet the cells by centrifuging at 3000xg at room temperature for 5 minutes.
*Remove the supernatant.
*Resuspend the cell pellet in 5 mL PBS.
*Re-pellet the cells by centrifuging at 3000xg at room temp for 5 min.
*Repeat wash with 5 mL PBS.
*Re-suspend washed cells in 0.5 mL PBS. The cell concentration should be approximately 5x10<sup>8</sup> cells/mL.
*Add 250 uL washed cells through the cell strainer lid into a 5 mL polystyrene tube. To get the solution to pass through the strainer, apply slight pressure to the strainer lid with the pipet tip as dispensing the cell solution.
*Add 250 uL PBS through the cell strainer lid into the 5 mL polystyrene tube.
*Put cells on ice.
*Analyze cells by flow cytometry using 488 nm excitation as quickly as possible.

Latest revision as of 15:06, 22 April 2008

FACS protocol V0.1

Materials

  • 5 promoter test strains provided as stabs from Endy Lab
    • <bbpart>I20259</bbpart>,<bbpart>I20260</bbpart>,<bbpart>I20268</bbpart>,<bbpart>I20269</bbpart>,<bbpart>I20270</bbpart>
  • TOP10 background strain (Jason will provide so we're all actually using the same one).
  • 100 mM sodium phosphate, 150 mM sodium chloride, pH 7.4 (PBS)
  • 5 mL polystyrene round bottom tube with cell strainer cap: BD Falcon #352235
    • Jason R. Kelly 13:37, 15 February 2008 (EST):What's the cell strainer cap for? Don't normally use that.
    • CAjoF 12:42, 18 February 2008 (EST): The cell strainer cap is make sure there are no big particulates in the sample that may clog the FACS. It is more important for fixed cells, but it certainly never hurts.
  • Supplemented M9 Media

Culturing of the strains

  1. Streak LB+Kan plates of the 5 test strains and an LB (no antibiotic) for TOP10
    • Julius B. Lucks 19:02, 3 April 2008 (EDT): This was done for us by CAjoF. Plates were stored in the 4C refrigerator for 2 weeks.
  2. For each test construct add 5ml of supplemented M9 medium (w/glycerol, see preparation protocol) and Kanamycin (20ug/ml) to three 17mm test tubes (so 15 tubes total). Add 5ml of supplemented M9 medium (w/glycerol) with no antibiotic for the TOP10 negative control cells to three 17mm test tubes.
    • how many replicates do we want to do (X)?
    • I would suggest 3 to start. If the replicates are wildly different, we can move to more.CAjoF 12:45, 18 February 2008 (EST)
  3. Inoculate media by picking single colonies from plates. Grow cultures for 20 hrs at 37°C with spinning at 70 rpm.
    • Kim de Mora I had trouble using culture sticks to innoulate the first time I did the experiment. There was a lot of debris that was picked up by the machine. Works much better with tips.
    • Dileep D. Monie 13:49, 1 April 2008 (EDT): 70 RPM seems a bit slow. Any reason why I shouldn't shake more vigorously?
      • Jason R. Kelly 14:47, 1 April 2008 (EDT): I think you should be OK using whatever RPM you normally use. The variability between shakers/rollers makes me guess we couldn't easily standardize this if we wanted to.
  4. In the morning dilute 100x into 5ml of pre-warmed (37C) fresh media to get cells back into log phase and let grow for 4hrs under same conditions as overnight
    • Does the back-dilution use media containing antibiotic for the 5 test strains? CAjoF 16:11, 29 February 2008 (EST)
  5. The cultures should be at an OD of 0.2 - 0.4 after 4hrs.

Live Cell FACS Procedure

  1. When your 5 mL culture reaches OD600 ~ 0.300, pellet the cells by centrifuging 1ml of each culture in an eppendorf at 3000xg at room temperature for 5 minutes.
    • Kim de Mora: I did this with an OD of 0.1 and with 250 μL of washed cells, still had 90 000 events/sec on our LSR-II.-
  2. Remove the supernatant.
  3. Resuspend the cell pellet in 1 mL PBS.
  4. Add 250 uL washed cells through the cell strainer lid into a 5 mL polystyrene tube. To get the solution to pass through the strainer, apply slight pressure to the strainer lid with the pipet tip as dispensing the cell solution.
  5. Add 250 uL PBS through the cell strainer lid into the 5 mL polystyrene tube.
  6. Place cells on ice.
  7. Analyze cells by flow cytometry using 488 nm excitation as quickly as possible.

Analysis of FACS Data

  1. Using the signal from the TOP10 cells, create a gate (P1, in image below) in the forward scatter-side scatter plot that captures the core of the scattering events.
  2. Using the signal from the TOP10 cells, create a second gate (P3, in image below) on the fluorescence channel which includes 99% of the total population and a third gate (P4, in images below) which encompasses all greater fluorescence signals.
  3. Copy these gates to all other samples.
  4. Determine the geometric mean of fluorescence intensity of the P4-gated population in each sample, <Isample,P4>.
    • Dileep D. Monie 18:38, 3 April 2008 (EDT):The geometric mean of I20259 should be calculated using the P3-gated population.
  5. Determine the geometric mean of the P3-gated population of the TOP10 cells, <ITOP10,P3>. This corresponds to the mean background fluorescence intensity of the cells.
  6. Calculate the fluorescence intensity in consensus Biobrick GFP units = I/II20260 = <Isample,P4>-<ITOP10,P3>/(<II20260,P4>-<ITOP10,P3>).
side scatter vs. forward scatter dot plot for TOP10 cells, showing gated populations corresponding to cells
Histogram of raw GFP fluorescence/cell for TOP10 cells, showing gates created with this negative control
Histogram of raw GFP fluorescence/cell for I20260 cells, showing copied gates.