Functionele Complementatie Analysis (FCA): A Laboratory Oefening ontworpen en geïmplementeerd om het onderwijs in Biochemical Pathways Supplement

The overall goal of the Functional Complementation Essay is to elucidate the activity of an enzyme by introducing a functional copy of the corresponding gene into a mutant cell to see if it restores a wild type phenotype in the mutant background. This method can help answer key questions in a variety of fields, such as biochemistry, microbiology, genetics, plant biology, evolution, and others. The main advantage of this technique is that it assesses the function, activity, and/or role of an enzyme, under in vivo conditions, which are normally physiological in nature, as opposed to in vitro conditions, which are normally artificial in nature.

The implications of this technique, extend towards identification, and/or the elucidation of the function of uncharacterized enzymes, and those that still have putative or predicted functions. Demonstrating this procedure of functional complementation is biotechnology and molecular biosciences major, Taylor Harkness, a student in my laboratory. After the cloning the DapL, TarB, MurE, and RSH genes according to the text protocol, inoculate 50 milliliters of liquid medium with a single colony of mutant bacterial cells to be transformed with a gene of interest and grow overnight.

On day two, use the 50 milliliter overnight culture to inoculate 1 liter of the appropriate medium and grow at 30 degrees Celsius to lock-phase, or to an OD600 of 0.4 to 0.6. Harvest the cells by centrifugation at 5, 000 times G and four degrees Celsius for 15 minutes. Then decant the supernatant and use 500 milliliters of sterile, ice cold, distilled water to resuspend the pellet.

Centrifuge the cells again, and after decanting the supernatant, use 250 milliliters of sterile, ice cold 10%glycerol to resuspend the pellet. After spinning the cells a final time, use two milliliters of 10%glycerol to resuspend the cells. Transfer 50 microliter aliquots into microcentrifuge tubes and immediately freeze by placing the tubes in a dry ice ethanol bath.

Store the cells at minus 80 degrees Celsius for electroporation. To carry out electroporation, add 1 microliter of recombinant plasmid to a 50 microliter aliquot of competent cells and place on ice for five minutes. Transfer the cells to an electroporation cuvette and set the electroporation apparatus to the following settings:25 degrees Fahrenheit capacitance, 2.5 kilovolts, and 200 ohm resistance.

Then with the cuvette in the device, deliver a pulse. Add 1 milliliter of recovery medium to the cuvette and transfer the cells to a 15 milliliter conical tube. Allow the cells to recover by incubating the culture in a shaking incubator with gentle rotation for 60 minutes at the appropriate temperature required by the mutant.

To select for transformants, pipette 100 microliters of the culture onto medium with agar plates and use a sterile spreader to spread the solution. Then, incubate overnight at the appropriate temperature. Refer to the text protocol for further details.

To perform complementation analysis, transform AOH1 with the empty vector pBAD33 or with dapL expression vectors using electroporation, as just demonstrated. Plate the transformation mixture on LB agar medium, supplemented with 50 micrograms per milliliter of Dap, 34 micrograms per milliliter of chloramphenicol, and 50 micrograms per milliliter of kanamycin. Incubate the plates at 30 degrees Celsius for 24 hours before observing the results.

With the empty vector pBAD33 or with the murE expressing vector, transform the TKL-11 mutant using electroporation as demonstrated earlier in this video. Plate the transformants on LB agar, supplemented with 50 micrograms per milliliter of thiamine, and 34 micrograms per milliliter of chloramphenicol, and incubate the plates at 30 degrees Celsius for 24 hours before checking for transformants. Test for complementation by streaking or plating colonies from both the control and experimental transformations onto two plates of LB medium, plus 0.2%arabinose, and 50 micrograms per milliliter of thiamine.

Incubate one plate at 30 degrees Celsius and the other at 42 degrees Celsius for 24 hours before assessing the growth phenotype. Transform the mutant Hx699 strain and the wild type Rr217 strain of novosphingobium species with the pRK290 empty vector, or a vector containing the native rshNsp gene in two separate transformation events each, as demonstrated earlier in this video. Plate the transformants on potato dextrose or PD agar supplemented with 10 micrograms per milliliter of tetracycline and incubate for at least 72 hours, or until transformants appear.

Then, streak the transformants in an X-pattern on fresh PD agar plates with tetracycline. After incubating at 30 degrees Celsius for at least four days, visually examine the growth phenotype of both plates. The E.coli double mutant AOH1 harbors a mutation in the DapE gene, and a deletion of the DapD gene, and won't grow unless Dap is provided.

As seen here, the AOH1 mutant expressing DapLs is able to grow on LLDap-free medium by converting THDP to LLDap in the Dap lyse pathway. The MurE enzyme facilitates the addition of m-DAP into the peptidoglycan of gram-negative bacteria. The E.coli MurE mutant, TKL-11, cannot grow at 42 degrees Celsius.

In this experiment, only TKL-11 cells transformed with MurE grow at 42 degrees Celsius. A mutation in the TyrB gene prevents the synthesis of tyrosine and phenylalanine. However, as shown here, when the TyrB mutant DL39 strain expresses the A.thaliana At5g36160 gene, it grows on M9 medium, lacking tyrosine and phenylalanine, demonstrating that the enzyme can synthesize tyrosine.

The Hx699 mutant in novosphingobium species harbors a non-functional RSH protein and produces hypomucoidal phenotype. However, transformation with the native RSH gene produces more soluble extracellular polysaccharides in the multicellular X-streak that is hypermucoidal. In contrast, when the Hx699 mutant is transformed with an empty vector, it produces a hypomucoidal phenotype.

Once mastered, this technique can be done in approximately 24 to 48 hours. If performed properly, depending on the growth of the model organism used, excluding the cloning, competence of preparation and transformation steps. While attempting this procedure, it is important to remember the growth condition requirements of the mutants used in the functional complementation analysis.

Following this procedure, other methods like traditional in-vitro axiomatic characterizations can be performed in order to answer additional questions like substrate specificity, temperature, and pH optima, and somatic velocity rates, et cetera. After its development, this technique paves the way for researchers in the field of biology and many subdivision fields, such as microbiology, to elucidate the in vivo function, or role, of enzymes from a variety of organisms. After watching this video, you should have a good understanding of how to make electrocompetent bacterial cells, how to transform bacteria using electroporation, and how to assess the function of genes/enzymes using functional complementation.

Don't forget that working with pathogenic organisms can be extremely hazardous and the necessary precautions should be taken.