Need Tips and Tricks on plate-reading Lycopene production of E.coli

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In summary, the conversation discusses the use of acetone to extract lycopene from bacteria, but the evaporation of the solvent has been causing issues with the results. Some literature suggests using HPLC for measurement, while others mention using absorbance but do not provide detailed methods. The individual is seeking advice on how to slow down evaporation or if a different solvent should be used. They also mention finding a solvent that can penetrate E.coli cell walls and has a higher boiling point than acetone. The possibility of refrigerating the samples is also mentioned.
  • #1
koitk
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TL;DR Summary
I need to measure the Lycopene production of E.coli strain on different mediums.
I have used Acetone, to extract Lycopene from bacteria, as described in literature. But when I started to measure the absorbance (470 nm) using 96-well microplates, some of the acetone would evaporate and ruin my results (even with the lid on).
Most of the literature that i have read uses HPLC to evaluate production, but some have used absorbance as well, but they don't specify their methods in great detail.
Can anyone help me by give me some tips, how could i slow down the evaporation. Or maybe i should use a different solvent?

Thank you in advance,
a desperate undergrad :)
 
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  • #3
Thank you for your reply!
This is a very detailed description of lycopene extraction from tomatoes. But it will not be of much use to me.
Extraction of lycopene form bacterium is much easier, and i have read articles about it. I just need to add solvent, incubate and centrifuge pellet.
My main concern is finding a organic solvent, that can penetrate trough E.coli cell walls, is transparent and has a higher boiling point than acetone (56 C).
Or are there any tips, how to use acetone as a solvent and read 96-well microplates in a plate reader, without it evaporating.
 
  • #4
Refrigerate the samples?
 

1. How do I set up my plate-reading experiment for measuring Lycopene production in E.coli?

To set up your experiment, you will need to prepare your E.coli culture and plate it onto agar plates. Once the bacteria has grown, you will need to add a solvent to extract the Lycopene. Then, use a spectrophotometer to measure the absorbance of the extracted Lycopene at a specific wavelength.

2. What is the best solvent to use for extracting Lycopene from E.coli?

The best solvent for extracting Lycopene from E.coli is a mixture of acetone and hexane. This solvent mixture has been found to efficiently extract Lycopene without damaging the bacteria.

3. How can I ensure accurate and consistent plate-reading results for Lycopene production in E.coli?

To ensure accurate and consistent results, it is important to maintain a controlled environment during the experiment. This includes using the same type of agar plates, solvent mixture, and spectrophotometer settings for each sample. It is also important to properly calibrate the spectrophotometer before each use.

4. What is the expected range of Lycopene production in E.coli and how can I interpret my results?

The expected range of Lycopene production in E.coli can vary depending on the strain of bacteria and the experimental conditions. It is best to compare your results to a control sample and calculate the percentage change in Lycopene production. Any significant increase or decrease in production can indicate the effectiveness of your experimental conditions.

5. Are there any troubleshooting tips for common issues with plate-reading Lycopene production in E.coli?

Some common issues with this experiment may include contamination of the agar plates, improper extraction of Lycopene, or inaccurate spectrophotometer readings. To troubleshoot these issues, it is important to properly sterilize equipment, use a proper solvent mixture, and double check calibration and settings on the spectrophotometer.

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