In this high throughput screen for fungal endo gluconate activity in e coli, the common microbial dye Congo Red is used to visualize enzymatic degradation of carboxy methyl cellulose by cells growing on solid medium first cells are transformed with an endo gluconate library and plated selectively for single colonies. Then individual clones are picked and grown overnight in 96 well plates. Next, an enzymatic screen is performed by replica plating on CMC plates containing IPTG to induce endo gluconate expression.
Finally, the act of clones are picked from the master plate stored at minus 80 degrees Celsius. This assay produces unambiguous results as zones of degradation at the colony site, where halo size correlates with enzymatic activity and allows qualitative assessment of relative protein fitness. The main advantage of this technique over existing methods like the overlay method, is that it allows high throughput analysis with a very low false positive rate.
Add 25 grams of LB growth medium, 15 grams of agar, and 1.5 grams of carboxy methylcellulose substrate to one liter of distilled water, an autoclave to sterilize. After the medium is cooled, add appropriate antibiotics and IPTG to a final concentration of 100 micromolar. Now pour 200 milliliters of medium each into five large square Petri dishes or bio essay trays, and allow plates to dry completely.
First, generate a library of endo gluconate catalytic domains into a suitable vector like PET 22 B plus from novagen. First, generate a library of endo gluconate catalytic domains into a suitable vector like PET 22 B plus from Novagen that contains the T seven promoter with a lack operator and a PAL LV signal sequence for targeting to the peri plasm. Transform the endo gluconate library into e coli strain BL 21 DE three plate for single colonies using glass beads and incubate the transformation overnight at 37 degrees Celsius using sterile toothpicks inoculate clones into 96.
Well deep well plates containing 400 microliters of LB mediums supplemented with appropriate antibiotics. Incubate at 37 degrees Celsius overnight with shaking at 250 RPM. Now add sterile glycerol to 96 well overnight cultures for long-term storage at minus 80 degrees Celsius.
This will be the master plate using a 96 pin stamp, replicate plate overnight cultures onto the screening plates containing IPTG and CMC for each screening plate. Stamp five sets of overnight cultures label and incubated room temperature until visible colonies have formed. Continue to rinse the plate with distilled water until all traces of the colonies are removed.
Pour just enough, 0.5%Congo red to cover the washed plate and incubate for a maximum of 15 minutes At room temperature. Pour off the dye at a generous amount of one molar sodium chloride and incubate 15 minutes at room temperature. Then discard the sodium chloride to reveal CMC degradation halos.
Using a sterile toothpick, select active enzymes from the master plate for further characterization. In this example, endo gluconate expression vectors were generated in PET 22 B plus under the control of the T seven promoter with a lack operator. The resulting T performance of endo gluconate expressing BL 21 colonies were then screened to produce CMC degradation halos After washing and development with Congo Red, typically clones on these screening plates can be identified as inactive, weekly active, and highly active based on the size of the degradation zones.
Here, the identification of active enzymes is robust and reproducible. It is important to remember to properly label the screening plates in order to correctly identify positive clones in the master plate.
