Colony-picking

A Petri dish containing bacterial colonies is blotted with nitrocellulose paper. This transfers a large portion of each colony to the paper, which is saturated with a solution that lyses (breaks open) the cells. The DNA of the lysed colonies is denatured with alkali. The nitrocellulose paper is neutralized, washed, and the paper either baked in an oven or treated with ultraviolet light to immobilize the denatured DNA. The DNA on the paper is hybridized with the labeled probe of interest, and the excess label is washed off. The dried paper is exposed to photographic film and the film developed. The exposed spots on the film can be matched with the colonies on the master plate and colonies picked off for further study. 

Directed evolution relies on the analysis of large numbers of clones to enable the discovery of rare variants with unproved function. In order to analyze these large libraries, methods of screening or selection have been developed, many of which use specialized equipment or automation. These range from the use of multichannel pipettes, all the way up to robotics, depending on the level of investment [59]. Specialized robotic systems are available to perform tasks such as colony picking, cell culture, protein purification, and cell-based assays. 

Colony-based ee assays have not been developed so far. Rather, all the assays described in this chapter are carried out in the wells of micro titer plates following colony picking. 

Fig. 8.2. Stages of enzyme screening. Microorganisms are grown in flasks or deep-well microplates. Separation of single clones is achieved either by FACS or by plating on agar plates and subsequent colony picking into microplates. Finally, assays are carried out in microplate formats using whole bacteria or cell extracts. Alternatively, bacterial clones can be directly screened on the level of colonies on indicator agar plates or during FACS.

Fig. 9.4 The colony lift screen. Step 1 Bacteria are spread on a Supor (low protein binding) filter on YTG -agar plate and grown for lOh at 30°C to form microcolonies. Step 2 The Supor filter is placed on top of cellulose-acetate filter on IPTG containing plate at 30°C for 16 h (induction of scFv-CBD expression). During the induction period the scFv-CBD fusion are secreted, diffuse and bind tightly to the cellulose acetate filter. Step 3 The colonies on Supor filter are saved for recovery later. The cellulose acetate filter is processed with labeled antigen as illustrated in the cartoon. Step 4 Probable binders are identified on the cellulose-acetate filter and colonies picked from the master Supor filter. These candidates are later verified by ELISA for specificity...

Positive colonies picked into 15-mL polypropylene tubes containing 3-4 mL LBamp can be grown up and plasmid DNA prepared using a variety of mmi-preparation protocols This DNA will be used m subsequent experiments as it represents a single, homogeneous, DNA fragment... 

The photograph shows the two ml standard deep well plate with 1.6 ml TAP-agar. Transformant colonies where picked from plate, spread inside the wells with plating-beads and incubated for 12 days at 20°C under illumination. The plate was afterwards sealed with a MicroMat ... 

In taking this drastic action, the bacterial cells collectively desert a locale where nutrients have become scarce. Now a packet of spores, they await transportation to a new home. The wind, an animal, or perhaps flowing water will pick up the fruiting body and deposit it elsewhere. The spores of course do not guide their journey, but if by chance they land in an appropriate environment, they then revert to their free-living form. If nourishment is plentiful, they may establish a flourishing new colony of bacteria. 

Pick positive colony from original plate... 

Pick colonies, grow, prepare plasmid. PCR screen for insert... 

Pick colonies and prepare expression-ready plasmids. 

Pick colonies for plasmid miniprepping as usual (if blue-white screening is used then blue colonies should constitute <10% if the reactions were successful. (The blue colonies are derived from inefficiently linearized parental plasmid and should not be picked as they are non- recombinant ). Two colonies are normally sufficient to find a recombinant clone but more may be required. 

Pick t ie colonies as indicated in subheading 3.1 from point 4 to point 9 and expand each clone in selection medium. 

Pick up a colony see Note 12) and check the plasmid by restriction enzyme digestion followed by agarose gel electrophoresis. 

---