Automated Selection and Isolation of Bacterial Colonies With the CellCelector in Biotechnological and Pharmaceutical Research
Bacteria are industrially used for the production of a number of enzymes and other organic molecules like alcohol. Also there is a huge interest in developing bacterial host systems for the production of antibodies and other pharmaceutical products because of enhanced yields and lower costs compared to the predominantly used mammalian host systems.
For the latter, the gene of the desired product is inserted into a plasmid, which is then used for transformation of bacteria. Though, not all plasmids transformed into bacteria might contain the insert. A common method to identify bacteria transformed with a plasmid bearing the gene of the desired product is the blue-white screening. Bacteria containing plasmids with successfully ligated inserts appear white, while bacteria with plasmids not containing the insert appear blue, which enables an easy detection of positive bacteria.
To reach a high throughput and optimal yields an automation of the production process is of great interest. The CellCelector enables the automated selection and isolation of positive bacterial colonies for evaluation and further downstream processing.
Isolation of Bacterial Colonies Workflow
Step 1: Scan and Imaging
Scanning process
Petri dish with bacterial colonies on the motorized stage (A);
Overview image after scanning (B);
Enlarged area in fluorescence illumination (C).
Step 2: Detection of Target Colonies
Colony detection by defining grey value thresholds
Graphical view of grey values (A) of the reference image (C). By moving the upper (blue bar) and lower (red bar) limit a range of grey values (green bar) can be defined and used for detection. Detected colonies on reference image (D); Detected Colonies at the total scanned area (overview image B).
Step 3: Automated Picking of Bacterial Colonies
Semi-solid media picking module - developed for picking cell colonies from semi liquid medium (e.g. Methylcellulose) and from solid media (e.g. Agar).
Picking tool right after taken up a fresh tip from a rack (left);
Picking tool performing a picking process within a fluorescence illuminated agar label plate with Deinococcus colonies (right).
Step 4: Documentation
Documentation of the isolation of a Deinococcus colony out of an agar plate. It is shown the region of a culture dish before (left) and after (right) the colony isolation process.
Application Example: Precise Isolation of Deinococcus Colonies Out of Agar Plates
Deinococcus is a genus of bacteria having high resistance to numerous kind of stress (radiation, oxidation, heat, drought, cold, solvents, alkaline, acid, ethanol, butanol etc.). Additionally to that this Bacterium has a short generation time (approximately 80 minutes) and the ability to utilize a vast range of sugars, amino and organic acids as a carbon source for catabolism which makes it interesting for industrial purposes. Moreover Deinococcus cells are able to integrate large fragments of DNA from bacteria, fungi and plants in a highly stable manner and so there are able to produce highly complex compounds like carotenoids, enzymes, antibiotics etc.
Summary of a picking experiment of Deinococcus colonies from agar plates
As example there are shown the images of five colonies (with the ID of 24, 119, 111, 57 and 53) before and after picking, the colonies within the destination well and the position of the colonies in the source plate.
The before and after pick images as well as the overview image of the entire PetriDish were taken and saved automatically by the CellCelector system.
The colony with the ID 24 is fluorescent.