Oct 16, 2021

Public workspaceMinimum inhibitory concentration of butanol for E. coli KJK01

DOI
dx.doi.org/10.17504/protocols.io.byz8px9w
  • 1in
  • iGEM IISER Pune India 2021
Arshshaikh
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DOI: dx.doi.org/10.17504/protocols.io.byz8px9w
Protocol Citation: Arshshaikh 2021. Minimum inhibitory concentration of butanol for E. coli KJK01. protocols.io https://dx.doi.org/10.17504/protocols.io.byz8px9w
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 13, 2021
Last Modified: October 16, 2021
Protocol Integer ID: 54048
Keywords: E. coli, Minimum inhibitory concentration, butanol
Abstract
This protocol helps you determine the minimum inhibitory concentration (MIC) of butanol for E. coli KJK01. However, this technique can be extended to determine the MIC of any metabolite for any strain by making adjustments to the concentration of the metabolite in the starting wells.
Take a 96 welled plate.

Media condition preparation
Media condition preparation
Calculate the amount of butanol that you require in the starting well. Wilbanks 2017 paper suggests that butanol shows strong toxic effects below 10 g/L and entirely inhibits growth at 15 g/L. We should expect our MIC to fall in this range. Hence, we can start with an initial concentration of 60 mg/ml.
CITATION
Wilbanks B, Trinh CT (2017). Comprehensive characterization of toxicity of fermentative metabolites on microbial growth.. Biotechnology for biofuels.
LINK
https://doi.org/10.1186/s13068-017-0952-4

Take Amount185.2 µL of LB and pour it in wells B2 to D2.

Pour Amount100 µL of LB of LB in all wells from B3 to B11 and the same for rows C and D.

Take Amount14.6 µL of Butanol and pour it wells B2 to D2 and mix well.

Take Amount100 µL of sample from well B2 and pour it in well B3. Mix well.

Take Amount100 µL of sample from well B3 and pour it in well B4. Mix well. Repeat this for the subsequent wells till you reach well B10.
Take Amount100 µL of sample from well B10 and discard in the well B12.
Note
Do not add the 100µL sample into well B11 as it is supposed to serve as our control (i.e. culture without butanol).


Repeat steps 5 to 7 for rows C and
Inoculation
Inoculation
Take Amount0.2 µL secondary overnight culture of wild type strain and inoculate wells B2 to B11.
Take Amount0.2 µL secondary overnight culture of uninduced KJK01 strain and inoculate wells C2 to C11.
Take Amount0.2 µL secondary overnight culture of induced KJK01 strain and inoculate wells D2 to D11.
Fill the border wells with milliQ.

Measurement
Measurement
Cover the 96 welled plate with foil and place it in the incubator at 37oC.
Take a reading of the plate using any plate reader every hour and place it back in the incubator. You may run this for 24 hrs.
Obtain a growth curve for each strain and concentration. Identify the least concentration for which the growth curve shows a sharp drop.
Citations
Step 2
Wilbanks B, Trinh CT. Comprehensive characterization of toxicity of fermentative metabolites on microbial growth.
https://doi.org/10.1186/s13068-017-0952-4