RECOMBINEERING INCREASES THE SPEED OF GENE CLONING
Assembling new DNA vectors with different genes of interest can become difficult when the gene is long, since it can be hard to identify unique restriction enzymes compatible with a polylinker that do not cut within the gene. Large recombinant DNA vectors can be created using homologous recombination, a process called recombineering (Fig. 3.25).
FIGURE 3.25 Recombineering
A gene of interest is flanked by sequences homologous to the BAC. Once inside the bacteria, the RED proteins recognize the ends of the gene of interest and facilitate homologous recombination between the BAC and gene of interest. The RED proteins are produced only when the bacteria are exposed to high temperatures, since their genes are controlled by a heat-inducible promoter.
To facilitate recombination, enzymes from lambda phage called RED are engineered to be expressed by a specific host strain of bacteria. They recognize homologous sequences and recombine them to form a single molecule. These proteins are so efficient that as little as a 45 base-pair region of homology is enough to initiate recombination. In practice, E. coli have the genes for the RED proteins under the control of a heat inducible promoter. The gene of interest is electroporated into bacteria that have the lambda RED proteins active in the cytoplasm. They recognize the ends of the gene of interest and find their homologous sequences. In this figure, the homologous sequences are found on the BAC, or bacterial artificial chromosome. The enzymes break the BAC at the appropriate location and add the gene of interest. The engineered BAC is removed from this strain of E. coli to prevent any residual RED proteins from initiating further recombination. Identifying which E. coli have the gene of interest is different for recombineering because the bacteria have the vector whether or not the insert recombines. Instead of using a positive selection scheme such as antibiotic resistance, a selection/counterselection scheme is used to identify the recombined vector containing the gene of interest (Fig. 3.26).
FIGURE 3.26 Selection and Counterselection in Recombineering
Recombineering vectors use a selection and counterselection method to identify which bacterium harbors the vector containing the gene of interest. In part A, the gene for galK encodes a galactose kinase. When bacteria expressing GalK are grown on 2-deoxygalactose (2-DOG), a toxin is produced, which kills the bacteria (top plate). The GalK also allows the bacteria to grow on galactose minimal media (bottom plate). In part B, recombineering replaces the galK gene with the gene of interest, and therefore, the bacteria can no longer grow on galactose minimal media (bottom plate). The lack of GalK allows the bacteria to grow on 2-DOG (top plate)
First, the vector contains a gene for galK, or galactose kinase, a gene essential for growth on galactose. The bacteria produce galactose kinase and are able to grow on minimal media that contains only galactose as a carbon source. GalK protein also converts 2-deoxygalactose (2-DOG) into a toxic substance, so bacteria expressing GalK die when grown on 2-DOG. After the recombination reaction occurs, the bacteria are plated onto minimal media that have only 2-deoxygalactose. If any bacteria still have galK, galactose kinase creates toxin from 2-deoxygalactose, and the bacteria die. When galK is replaced with the gene of interest, there is no toxin produced, and the bacteria grow.