This method can help answer key questions in the protein based research such as annotation of new proteins or protein domains, characterization of structural and functional properties of known proteins, definition of protein interaction networks or antigen antibody signatures in different conditions. The main advantage of this technique is that it is completely unbiased, high throughput and modular for any kind of study ranging from a single gene up to a whole genome. The construction of a domainone library is very useful for functional studies.
It can be transferred to a phage display context and used for the selection against different targets such as binding proteins or purified antibodies. The coupling with NGS enables an extremely sensitive and powerful analysis. At the same time, the web tools specifically developed gives a precise characterization of the whole domainome and interactome.
To construct the ORF library first, sonicate the DNA with 30 second pulses at 100%output. After sonication, load the ladder and the sonicated DNA on a 1.5%agarose gel. Then start the electrophoresis unit at five volts per centimeter for 15 minutes.
The agarose gel electrophoresis shows the presence of DNA smear confirming sonication. This is in comparison to the solid band obtained from a DNA not subjected to sonication. Then cut the gel portion with the fragmented DNA smear and purify using column based gel extraction kit.
Measure the concentration of the purified DNA in the UV spectrophotometer. Then follow the manufacturer's instructions and treat five micrograms of the insert with one microliter of quick blunting enzyme mix. Then inactivate the enzyme mix at 70 degrees Celsius for 10 minutes.
To prepare the filtering vector, first digest five micrograms of the purified cloning vector with EcoRV restriction enzyme. Then load the digested and undigested vectors and the molecular marker on a 1%agarose gel. Then heat inactivate the restriction enzyme at 80 degrees Celsius for 20 minutes.
Next, phosphorylate the digested vector with five units of phosphatase enzyme and 1/10 volume of the 10X phosphatase buffer. Then incubate the mixture at 37 degrees Celsius for 15 minutes. Then inactivate the phosphatase enzyme at 65 degrees Celsius for five minutes.
Next, extract the plasmid from the gel and purify the DNA. Then measure the concentration of the DNA in the UV spectrophotometer. To perform the ligation reaction, add 400 nanograms of the phosphorylated inserts to one microgram of digested vector, 10X T4 DNA ligase buffer and T4 DNA ligase to a final volume of 100 microliters.
Then incubate the ligation reaction at 16 degrees Celsius for overnight. The next day, heat inactivate the ligase at 65 degrees Celsius for 10 minutes. Next, add 1/10 volume of three molar sodium acetate at pH 5.2 and 2.5 volumes of 100%ethanol to precipitate the ligation product.
To precipitate DNA, mix the reagents and freeze at minus 80 degrees Celsius for 20 minutes. After 20 minutes, centrifuge at the highest speed for 20 minutes at four degrees Celsius. After centrifugation, expel the supernatant.
To the pellet, add 500 microliters of cold 70%ethanol and again centrifuge. Discard the supernatant at the end of the centrifugation. Once the pellet is air dried, dissolve the precipitated DNA in 10 microliters of water.
Before performing electroporation, arrange for the appropriate number of microcentrifuge tubes and 0.1 centimeter electroporation cuvettes on ice. Then add one microliter of the purified ligation product to 25 microliters of the cells. Then pipette the DNA, cell mixture to the cold electroporation cuvette and flick the tube.
Then wipe the water from the exterior of the cuvette and place in the electroporation unit and hit pulse. After the electroporation is over, quickly add one milliliter of liquid 2X YT medium without any antibiotic to the cells in the cuvette. Then transfer the cellular mixture to a 10 milliliter tube.
Leave the tube on a shaker at 220 revolutions per minute at 37 degrees Celsius for an hour. Plate the dilutions of the library on 10 centimeter 2X YT agar plates supplemented with chloramphenicol, ampicillin and only chloramphenicol. This is to obtain single colonies to be tested by PCR and to perform titration.
Next, plate the transformed competent cells on 15 centimeter 2X YT agar plates supplemented with chloramphenicol and ampicillin. Then incubate the plates at 30 degrees Celsius for overnight. The next day, count the colonies choosing the dilution where they are countable.
Then calculate the library size. Library size is calculated as follows, mean colony number times dilution factor times total library volume. Library size should be in the order of 10 to the power of six clones.
If ampicillin concentration is optimal to perform more filtering, the clone number reduces to one to 20 of that obtained in the absence of this antibiotic. To test for the positive colonies, use a tip to pick up around 15 to 20 single colonies. Then dilute the colonies with 100 microliters of 2X YT medium without antibiotics.
Next, PCR amplify 0.5 microliter of the culture as DNA template with Taq DNA polymerase. During the PCR, run 25 cycles of amplifications using annealing temperature of 55 degrees Celsius and an extension time of 40 seconds at 72 degrees Celsius. Check that the length of the insert is in the expected range of 150 to 750 base pair.
And that different colonies present different inserts with different size. This indicate a good library preparation in terms of variability. Next, add three milliliters of fresh 2X YT medium to the 150 millimeter plates to collect the bacteria.
Then harvest the bacteria using a sterile scraper. After mixing thoroughly, add 20%glycerol and leave in minus 80 degrees Celsius in aliquots for future use. For immediate use, perform column based plasmid extraction kit to purify the plasmid DNA from one of the aliquots of the library before adding glycerol.
Measure the concentration of the DNA in the UV spectrophotometer and then store the samples at minus 20 degrees Celsius until use. Use 2.5 microliters of the library as DNA template for PCR amplification. Set the thermocycler conditions and start the run.
Next, use an index PCR kit to perform index PCR. This will sequence the double indexed libraries within multiplexed Illumina runs. Then set the thermocycler conditions and start the run.
After purification with AMPure XP beads, to verify the size, run one microliter of the one to 10 dilution of the final library on the bioanalyzer. Then quantify by selecting the region of the final library trace. These schematic representations demonstate that after cloning into P filter vector, only colonies corresponding to ORFs produce functional beta-lactamase in the presence of antibiotics.
After filtering a decrease in the clone number of 20-fold is expected. With good-folder ORFs growing at higher antibiotic concentrations. Moreover, the ORF fragments can be easily recovered from the filtered library.
A phagemid ORF library is constructed to perform phage display selection against target proteins or antibodies. All the libraries and the outputs obtained are then deeply analyzed by the NGS. The NGS provides a complete information on library diversity, abundance and precise mapping of each of the selected fragments.
Finally the interactome seek webtool developed generates raw sequencing reads ranging to the list of putative domains with genomic annotations. After watching this video, you should have a good understanding of how to construct and validate a domainome library from a desired DNA source. And to perform a high throughput screening of the library by next generation sequencing.
We have optimized the sequencing of the ORF filtering libraries with the Illumina platforms and developed a webtool which make possible the analysis of this kind of data without any need of programming skills.
