Oct 14, 2019
Growth curve analysis
- 1Wageningen University
- iGEM Wageningen 2019
Protocol Citation: Sebastiaan Kuiper 2019. Growth curve analysis. protocols.io https://dx.doi.org/10.17504/protocols.io.77jhrkn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 14, 2019
Last Modified: October 14, 2019
Protocol Integer ID: 28619
Keywords: Growth curve analyses, Bacteriophages, Defense mechanisms, CRISPR, CRISPR-Cas, Cas9, Cpf1, Cas12a
Abstract
To observe the potential of defense mechanisms of either native or synthetic systems in Escherichia coli (and more) when incubated with a bacteriophage stock.
Materials
MATERIALS
96-well plate, flat bottom, tissue culture treated, black wall with clear bottomFisher ScientificCatalog #3904
Microplate Reader Synergy Mx
STEP MATERIALS
96-well plate, flat bottom, tissue culture treated, black wall with clear bottomFisher ScientificCatalog #3904
Protocol materials
96-well plate, flat bottom, tissue culture treated, black wall with clear bottomFisher ScientificCatalog #3904
Microplate Reader Synergy Mx
96-well plate, flat bottom, tissue culture treated, black wall with clear bottomFisher ScientificCatalog #3904
96-well plate, flat bottom, tissue culture treated, black wall with clear bottomFisher ScientificCatalog #3904
Preparations
Preparations
Media and bacteriophage stock solutions :
- 1L Luria-Bertani (LB) media (with antibiotics)
- Desired Bacteriophage stock solution in LB media (with known Plaque Forming Units (PFU) ml-1)
Fill in plate reader protocol as follows:
- Set temperature: 37°C
preheat before moving to next step
- Start kinetics:
Runtime 15:00:00 (HH:MM:SS), Interval 0:04:00
- Shake:
medium, 0:30 (MM:SS)
- Read:
Absorbance Endpoint, Full Plate
Wavelengths: 600
Read Speed: Normal, Delay: 100 msec
- End kinetics
Prepare overnight cultures of desired samples (with associated antibiotics).
Plate reader
Plate reader
Measure OD600 of overnight cultures and dilute cultures to an OD600 of 0.02
Load 180 µL of diluted overnight culture into a
96-well plate, flat bottom, tissue culture treated, black wall with clear bottomFisher ScientificCatalog #3904
Include a serie of LB (without bacteria) as a control and as zero point for the OD600 measurements!
Start plate reader protocol go to step #2 and let the bacteria grow to an OD600 of 0.11.
Prepare Bacteriophage PFU dilutions (with associated antibiotics) for;
MOI 101 : 4.0 x 1010 PFU ml-1
MOI 100 : 4.0 x 109 PFU ml-1
MOI 10-1 : 8.0 x 108 PFU ml-1
MOI 10-2 : 8.0 x 107 PFU ml-1
MOI 10-3 : 8.0 x 106 PFU ml-1
An OD600 of 0.10 correlates to 8.0 x 108 cells per ml.
The above concentrations are required when 20 μl of bacteriophage dilution is added into 180 μl of cell culture with an OD600 of 0.11 (1:10 dilution).
At the moment an OD600 of 0.11 is reached, the plate reader must be stopped and 20 µL of bacteriophage dilution* must be added to a final volume of 200 µL to both the samples and the LB controls.
* include as a control, a serie without bacteriophages and only LB media (with antibiotics)
Restart the plate reader protocol and measure over 15 hours the growth of the samples.