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Adenovirus Construction by Homologous Recombination in Bacteria


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Adenovirus Construction by Homologous Recombination in Bacteria

Adenovirus Construction by Homologous Recombination in Bacteria

Cloning a gene of interest by homologous recombination in bacteria is a two-step procedure. Firstly, the gene of interest is cloned into a shuttle plasmid vector by using standard molecular biology methods. The shuttle plasmid contains two fragments of adenovirus genomic sequence flanking the multicloning site. The presence and orientation of the gene of interest is confirmed by restriction digestion analysis and sequence analysis. Next, introduce the gene of interest into the adenovirus genome by homologous recombination between the shuttle plasmid and a large backbone plasmid. This backbone plasmid contains most of the adenovirus genome, however lacks essential genes for virus propagation, generally, E1 genes. Rapid detection of positive recombinants is achieved by antibiotic selection and restriction digestion analysis.

In our protocol, the recombination between the shuttle plasmid and the adenovirus genome contained in the backbone plasmid is performed in the E. coli strain. Positive recombinants are selected by resistance to an antibiotic.

Homologous Recombination in Bacteria

  1. Linearize the backbone plasmid with a restriction enzyme cutting in the insertion site, such as Swa I. Check background by transforming E. coli strain BJ5183 with 100 ng of digested plasmid.
  2. If the number of colonies obtained is higher than five, repeat steps 1. After verification, store in 200 μL aliquots.
  3. Digest 3 μg of the shuttle plasmid with two appropriate restriction enzymes. Use 10 U of each enzyme and allow the digestion to proceed overnight.
  4. Confirm complete digestion by agarose gel electrophoresis (1 %). Purify the DNA fragment containing the expression cassette using a commercial DNA fragment purification kit. Quantify the recovered DNA through measuring absorbance at 260 nm.
  5. Mix gently 50 ng of linearized pKP1.4 plasmid with different amounts of the purified DNA fragment. Start with the following molar ratios: 1:5 pKP:fragment (approx. 50 ng pKP: 50 ng of fragment) or 1:20 pKP:fragment (approx. 50 ng pKP: 200 ng of fragment).
  6. Transform competent E. coli strain BJ5183 by using standard procedures. Plate co-transformed bacteria in LB + Ampicillin dishes. Incubate at 37 °C overnight.
  7. Pick at least 12 isolated small colonies. Inoculate each in 3 mL of LB + Ampicillin. Incubate at 37 °C with shaking at 220 rpm overnight.
  8. Purify plasmid DNA with a commercial kit. Resuspend DNA in 30 μl of Milli-Q H2O. Check by agarose gel electrophoresis (1 %) and Store at −20 °C.
  9. Transform competent E. coli (strain DH5α, TOP10 or similar).
  10. Culture co-transformed bacteria in LB + Ampicillin dishes. Incubate overnight at 37 °C. Pick two colonies from each plate presenting less than 1,000 colonies and inoculate separately in 3 mL LB + Ampicillin. Grow overnight at 37 °C with shaking at 220 rpm.
  11. Purify plasmid DNA with a commercial DNA minipreparation kit and store at −20 °C. Proceed to check and identify positive recombinants with restriction enzymes.

Identity of the Vector Genome by Restriction Enzyme Analysis

  1. Digest 1 μg purified plasmid DNA from the colonies selected after homologous recombination with 0.3-0.5 U of an informative restriction enzyme, for 4 hours.
  2. Perform digestions with at least 7-8 different enzymes and run in a 1 % agarose gel.
  3. If one restriction enzyme pattern does not correspond with the expected pattern, repeat the digestion. If the observed pattern still does not correspond with the expected pattern or if there are more than one unexpected enzyme patterns, discard the selected DNA.
  4. Inoculate one positive recombinant in 200 mL LB + Ampicillin. Purify plasmid DNA with a commercial DNA maxipreparation kit and store at −20 °C.

Reference:

  1. Miravet S, et al. Construction, Production, and Purification of Recombinant Adenovirus Vectors. Methods in molecular biology (Clifton, N.J.), 2014, 1089:159-173.

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