This video demonstrates a procedure called, Chimera Assembly by Plasmid Recovery and Restriction Enzyme Site Insertion or CAPRRESI, in which two chimeric genes are generated by introducing specific restriction enzyme sites into synonym DNA and then, cutting and swapping the DNA sequences. In CAPRRESI, the wild type genes of interest are first inserted into the multiple cloning sites of the same vector. Next, break regions for each gene with the same amino acid sequence are selected and the break region for the Ampicillin resistance gene is selected.
Perl scripts are used to determine synonym sequences containing restriction sites for each break region. Using specially designed oligos, PCR is performed to produce four complementary parts that contain synonym DNA with the desired restriction enzyme sites. The resulting PCR products are purified, digested and then, ligated to product a chimeric sequence.
The main advantage of this technique is that it facilitates gene fusions. This method can help answer key questions in the molecular biology field such as, what are the functions of our proteinzyme. Begin this procedure with the genes of interest already inserted into the multiple cloning sites of the vectors.
Here, the rpoD and sigA genes, which encode bacteria signal factors needed to initiate RNA synthesis, have each been cloned into pUC18 as detailed in the accompanying text. To design the oligonucleotide for producing the chimeric protein, first, run the translate. pl perl script to obtain the amino acid sequence of rpoD, sigA and the ampR genes.
Then, using the muscle web server, align the amino acid sequences of rpoD and sigA. For each sequence, select a break region with the same three to six amino acid sequence. Save the sequences in the file, regions.fas.
Next, select a three to six amino acid long sequence inside the ampR gene, save this sequence in the file, regions.fas. To determine all the synonym DNA sequences, run the perl script, synonym.pl. Then, to find which restriction enzymes cut the synonym sequences, run the script, resynonym.pl.
Next, use the synonym DNA sequences to design oligonucleotides that incorporate the same restriction enzyme site into the break regions of rpoD and sigA. Design an oligonucleotide to disrupt the ampR gene that incorporates a different restriction enzyme site. The forward and reverse oligonucleotides will overlap at the restriction enzyme site.
Oligonucleotides should range from 21 to 27 nucleotides and with the cytozine or guanine and contain a restriction enzyme site. A forward oligonucleotide has the same sequence as its target region on the template DNA. A reverse oligonucleotide is the reverse complementary sequence of the target region on the template DNA.
Order the oligos from a DNA synthesis provider. When the oligonucleotides arrive, use them to perform PCR using the rpoD and sigA pUC18 constructs as template DNA. This will produce two complementary parts for each construction.
Following PCR, perform agarose gel electrophoresis to separate the PCR products from the DNA template. Purification of complementary plasmid parts is crucial to avoid parental plasmic contamination. The parental plasmids can be eliminated by gel purification as we are demonstrating here or, by dpn1 restriction enzyme digestion which can be used to chop up the parental plasmids.
After electrophoresis, stain the gel with ethidium bromide solution. Visualize the bends by placing the stained gel in a UV chamber at 300 nanometer. Keep the exposure of the gel to a minimum.
Then cut out the bends containing the PCR products and place them in micro-centrifuge tubes. Use a kit to purify the DNA samples, then using the appropriate fast digest restriction enzymes, perform a double digest on all of the complementary DNA fragments. AFL2 and SPE1 are used in order to fuse plasmid complementary parts.
Incubate the digestion reaction at 37 degree celsius for 15 minutes. Then, inactivate the restriction enzymes according to the manufacturer's guidelines. For example, inactivate AFL2, SPE1 at 80 degree celsius for 20 minutes.
Next, combine the appropriate DNA fragments with ligation buffer, water and T4 DNA ligase. Leave the ligation reaction at 25 degrees celsius for 10 minutes. In this way, the integrity of the ampR gene is restored, producing a functional protein.
Following the ligation, transform E.Coli DH5 alpha with three to five microliters of the chimera assembly ligation reaction. Plate the transformant cells in LB ampicillin x-gal IPTG plates and incubate them overnight at 37 degree celsius. The next day, select white colored transformant E.Coli colonies and streak them onto a new LB ampicillin plate.
Incubate the plates overnight at 37 degree celsius. To choose candidates for sequencing, perform colony PCR. Visualize the bends on a one to one 2%agarose gel by performing electrophoresis as before.
Select candidate colonies that display the DNA bend of the correct size to inoculate cultures. The next day, extract the plasmid DNA from them, using a kit. After plasmid purification, send the samples to be sequenced.
rpoD sigA chimeric constructs were generated as detailed in this video. Following sequencing, alignment of wild type and chimeric genes was performed using muscle software. The chimeric gene sequences were then assembled with mura software as shown here.
Synonym sequences appear in black and the AFL2 site appears underlined. This sequencing data demonstrates successful use of the CAPRRESI method to generate chimeric constructs. After watching this video, you should have a good option for fusing gene segments.
