The overall goal of this procedure is to create a random mutant library for a given target DNA sequence, and to rapidly identify and characterize mutants using semi-quantitative functional selection. This is accomplished by first transforming a plasmid with a coli, one origin of replication containing the target sequence into the mutator strain. After plating and incubating the cells overnight, they're harvested and the target plasmid is isolated to obtain the mutant library, the mutant library is then transformed into a readout strain to characterize the mutations in the second portion of the protocol, A gradient growth plate is constructed and bacterial cultures of the readout strain are loaded into a separate Petri dish containing soft aer.
The final step of the procedure is to stamp transfer these bacterial cultures to the gradient. Ultimately, results can be obtained that show changes in the phenotype profile of a given target sequence through differences in growth on a drug gradient. This video presents a method for the directed evolution of proteins in e coli.
This process mimics the natural evolution in the generation of random nutrient libraries, followed by a selection that constraints that diversity. Thus, thus, it improves our ability to generate new biochemical activities for industrial or biomedical purposes. This procedure has two components, the generation of random mutant library and the functional selection to obtain gain of function mutations demonstrating this procedure will be Jennifer Allen, a technician from my laboratory.
Prior to the start of this protocol thought electro competent JS 200 cells expressing a low fidelity variant of DNA polymerase one or low fidelity pole, one that had been previously prepared as described in the written protocol, these cells contain a temperature sensitive allele of pole one, such that low fidelity pole one activity predominates a 37 degrees Celsius, construct. A target plasmid containing a coli, one origin of replication with the target sequence cloned adjacent to the origin of replication, low fidelity pole one initiates coli, one plasmid replication. Thus introducing mutations to initiate mutagenesis.
Combine 40 microliters of electro competent cells and between 30 to 250 nanograms of target plasma DNA in a two millimeter gap. Electroporation vet pulse, the mixture in the electro at 1800 volts. Check the time constant or TC to ensure uniform ation conditions for each sample.
Ideally TC equals five to six milliseconds. Allow the cell DNA mixture to recover in one milliliter of LB broth for 40 minutes at 37 to Celsius shaking at 250 RPM. Obtain a 100 millimeter LV agro Petri dish prewarm to 37 degrees Celsius containing appropriate antibiotics to select for both plasmids plate the recovered cells at an appropriate dilution to obtain a near lawn concentration, which is defined as a distinct but uncountable number of colonies as shown in this example.
Following overnight incubation, harvest the bacterial colonies of the Petri dishes with LB broth. Isolate plasmid, DNA from the harvested cells, the resulting plasmid. DNA constitutes the random mutant library.
Perform a restriction digest by cutting the plasmid library with a restriction enzyme that linearizes both the target plasmid and the pole. One plasmid run an analytical aro gel on the isolated plasmid DNA to confirm quality and quantity. Once plasmids are verified, digest one microgram of the library with a restriction enzyme that linearizes the pole one plasmid, but does not cut the target plasmid.
After the restriction digest is complete, clean up the library using a DNA purification kit. Finally transform the clean library into a readout strain to characterize the mutations. To begin gradient preparation, mark 10 lanes spaced evenly across one edge of the bottom of the square petri dish.
Place the dish on an incline such that the bottom marked edge is elevated seven millimeters. Pour 25 milliliters of warm lb agar mixed well with an appropriate concentration of the selecting agent into the incline dish. This is the bottom layer of the gradient.
Make sure the LB agar coats the petri dish such that the elevated marked end of the dish contains approximately one millimeter of LB agar, and the lowered part contains about eight millimeters. Cover the plate with the lid KU for ventilation and allow the agri to set for 10 to 15 minutes. After the bottom layer of LB agar has hardened, move the dish to a flat surface.
Next, pour 25 milliliters of warm lb agar without the selecting agent to overlay the first agri surface, making sure to cover the entire bottom layer. Cover the plate with the lid KU for ventilation, and then allow the agri to set for 10 to 15 minutes to perform the stamp transfer of bacteria. First, obtain bacterial cultures of the readout strain and dilute them to have identical cell densities with an optical density at 600 nanometers of less than 1.0.
Next, transfer two milliliters of liquid soft agar equilibrated to 42 degrees celsius to the bottom of a 100 millimeter round Petri dish, pipette 40 microliters of the bacterial culture into the softer and then mix by rocking the plate. Coat the long edge of the glass microscope slide with the softer mixture. Then aligning the coated edge of the slide with the bottom mark on the gradient dish.
Touch the slide to the surface of the agar. A soft touch is sufficient to transfer a ribbon of the soft agar to the gradient surface. The slide is then set aside for cleaning and reuse.
Repeat this process. The remaining samples, experimental controls should be included on each gradient dish. If multiple gradients are being run, incubate the gradient dish top down overnight at 37 degrees Celsius times and temperatures of incubation may differ for different readout bacterial strains, but overnight at 37 degrees Celsius is typically sufficient for visible growth.
Finally, image and analyze the cell growth as described in the written protocol.Shown. Here are images of a readout strain transformed with either unmuted ized P-G-F-U-V or a PG FPUV mutant library that were carried through. Four rounds of mutagenesis dark or dim colonies indicate plasmid containing GFP inactivating mutations.
This figure represents the frequency of mutations throughout the neutral sequence of the target plasmid at 100 base pair intervals. Note the mutations occur across the entire plasmid sequence, but at the highest frequency nearest the origin of replication. Here, growth of mutants is shown against a drug gradient.
The distance grown toward the top of the gradient indicates differential drug resistance profile among the mutants. In this example, plasmid libraries of the human oxidative demethylase ABH two were screened for mutants with increased resistance to methyl methane sulfonate using drug selection gradients.Two. Such libraries representing two and four iterations of the mutagenesis protocol are shown in comparison to the parental wild type and the empty vector.
The white line denotes the threshold above which individual mutant colonies were isolated. For further phenotypic analysis, beta-lactamase mutants, R 1 64 H and E 1 0 4 KR 1 64 HG 2 67 R previously identified using low fidelity P one mutagenesis and astri and m selection are shown on a 0.4 microgram per milliliter and a four microgram per milliliter. Cefotaxime gradient protection against cefotaxime is indicative of extended spectrum beta-lactamase activity.
Note that the wild type beta-lactamase enzyme confers no protection relative to cells expressing an empty vector. Therefore, these mutants represent adaptation or the evolution of a new biochemical activity. Here we have presented a powerful combination of mutagenesis and functional selection for this generation of new biochemical activities.
A functional selection can be obtained either through drug selection or through genetic complementation, and it capitalizes on our ability to generate large genetic diversity. Pathogenesis may have to be restricted to a given sequence in order to optimize preexisting protein activities or for site directed pathogenesis. These can be easily done by cloning.
In addition, mutagenesis can be decoupled from a functional selection in order to obtain signature attacks or mutational footprints.
