Use of In Vivo Assembly for High-efficiency Plasmid Construction

My doctoral research is focused on studying proteins that are secreted by bacteria. I specifically want to understand if some of these secreted proteins are important for causing human infections. A major challenge in working with recently isolated clinical bacteria is their antibiotic resistance, which hinders molecular genetic techniques that rely on antibiotic selection, such as gene deletions and genetic complementation.

This cloning protocol is faster and more cost effective since IVA eliminates the need for specialized enzymes and sequential enzymatic reactions for plasmid assembly before E.coli transformation. To begin, launch a specialized DNA analysis software on a computer system. Artificially assemble the desired plasmid.

Then design primers that bind to each DNA fragment. Combine DNA isolated using commercial kits with the primers containing homologous DNA in a PCR tube. Then amplify the DNA with a PCR run.

Once the PCR reaction is complete, pipette out a two microliter aliquot. Combine it with loading dye. Perform agarose gel electrophoresis on a 1%agarose gel to separate DNA fragments.

Then use an ultraviolet or LED trans illuminator to visualize the fragments. Next, add one microliter of DpnI directly to the PCR reaction tube to remove methylated template DNA. Incubate the reaction at 37 degrees Celsius for 15 minutes or overnight.

With a nucleic acid purification kit remove residual enzymes, salts, primer dimers, and low molecular weight DNA products. Quantify the DNA concentration of the purified fragments using spectro photometry. Obtain pure amplified plasmid DNA.

Calculate the amount of plasmid and insert DNA required for each in vivo assembly reaction. Now, combine the calculated volumes of plasmid and insert DNA into a pre chilled 1.5 milliliter micro centrifuge tube. Place the tube on ice.

Next, pipette 25 to 100 microliters of thawed recA-chemically competent Escherichia coli into a tube. Transfer the DNA mixture into the aliquot. To perform heat shock transformation first incubate the mixture of E.coli and DNA on ice for 30 minutes.

Transfer the tube to a 42 degrees Celsius water bath for one minute. Then transfer it back on ice for two minutes. Aseptically transfer LB media to the tube to make up the volume to one milliliter.

Transfer the one milliliter mixture to a glass culture tube. Then place it in a shaking incubator for recovery and production of the plasmid encoded antibiotic selection marker. After recovery, deposit about 100 to 1, 000 microliters of the transformation reaction onto solid selection media.

Use a sterile cell spreader to disperse the aliquot across the auger culture plate. Centrifuge the remaining transformed cells at 13, 000 G for one minute. Then discard the supernatant and re-suspend the cell pellet in 100 microliters of sterile media.

Deposit the re-suspended cells onto solid selection media as demonstrated earlier. Then invert the culture plates. Place them in an incubator overnight at 37 degrees Celsius.

Remove culture plates containing transformed E.coli cultures from the incubator. Count the colonies directly to enumerate. Transfer sterile growth media supplemented with appropriate antibiotics into sterile culture tubes.

With a sterile transfer loop pick up a colony and inoculate the cells into the culture media. Incubate the culture tubes in a shaking incubator. The next day, isolate plasmid DNA from the bacterial cultures using a commercially available plasmid isolation kit.

To determine if the plasmids are correctly assembled use spectro photometry to quantify the concentration of DNA. Pipette out a 50 to 150 nanogram aliquot of plasmid DNA for a diagnostic restriction digestion reaction. Add restriction enzymes selected based on their expected cleavage sites on the plasmid DNA.

Once the restriction reaction is complete add loading dye to the tube. Load the entire mixture onto a 1%agarose gel, and perform an electrophoretic run. After the run, visualize the DNA fragments with a UV or LED trans illuminator for comparison against the latter.

Enzymatic digestion of two isolated plasmid clones resulted in a single DNA product of 2.3 kilobase pairs for Plasmid clone 1, indicating that this is PSU 19 alone. Single cleavage of Plasmid clone 2 resulted in a single three kilobase pairs DNA product, and two DNA products of 2.3 kilobase pairs and 770 kilobase pairs after double cleavage.