This article details a method for culturing genetically modified bacteria that express a fluorescent protein and confer antibiotic resistance. The process involves inoculating the bacteria into a nutrient medium, monitoring growth, and analyzing protein secretion using an automated system.
Take the culture of a genetically modified bacterium engineered to harbor a plasmid that expresses a secretory fluorescent protein and confers antibiotic resistance.
Inoculate it into a medium containing the antibiotic and incubate with shaking to promote bacterial growth.
The antibiotic ensures that only cells retaining the plasmid can proliferate.
Once the culture reaches the desired density, transfer it to an automated liquid-handling system.
A deep-well plate contains varying concentrations of essential nutrients, which are dispensed into a microtiter plate to create distinct media formulations.
Inoculate each well of the plate with the bacterial culture.
Seal the plate and place it into a microbioreactor, which maintains uniform temperature and aeration.
The different formulations lead to variations in bacterial growth and fluorescent protein secretion.
An optical sensor in the device measures cell density and fluorescence, enabling comparison of bacterial growth and fluorescent protein production across the media formulations.
Sterilize deep well plates for stock solution storage on a work table by wiping the plates with 70 percent ethanol and subsequently drying them in a laminar flow hood. Start the seed culture by inoculating 50 milliliters of BHI medium containing 25 milligrams per liter of kanamycin with one aliquot from the working cell bank. Place all necessary labware on the robotic work table and pour stock solutions in the corresponding labware.
Then, start the robotic work flow for media preparation so that the last step, which is the inoculation, is reached in time for the start of seed culture. Sample the seed culture after approximately two hours to monitor the growth by measuring the optical density at 600 nanometers or OD600. Continue to monitor the growth at each hour.
After approximately five hours, the culture reaches an OD600 between three and four, and is used to inoculate the main cultures. Place the seed culture on the liquid handler work table and continue the media preparation protocol. Seal the cultivation microtiter plates after inoculation.
Next, place the sealed cultivation MTP in the Biolector device. Then, start the predefined cultivation protocol.
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