1/1/2024 0 Comments Crossout pyrePR01 with the enolase gene substituted with the E. PR01 with the RNase J2 gene deleted (between coordinates 12687394) using the pRLYE1 vector PR01 with the RNase Y gene deleted (between coordinates 12819492) using the pRLYE1 vector PR01 with the RNase J1 gene deleted (between coordinates 10668378) using the pRLYE1 vector SA564RD with the pRLBER9 plasmid recombined into the chromosome, thereby disrupting pyrE expression SA564 hsdR type III mutant (restriction deficient) ![]() Restriction deficient, modification proficient Additionally, alternative origins would also be desirable, since both pMAD and pKOR1 use the pE194ts origin of replication.ĭescription, relevant genotype/characteristic, or purpose a A further complication with pKOR1 is the lack of alternatives to the chloramphenicol resistance marker on the backbone vector, which prevents its use in strains that are already resistant to this drug. aureus, such as strain COL or our clinical strain S30 ( 11, 24), carry the pT181 plasmid that carries the gene that encodes a tetracycline efflux pump, which may limit the use of the inducible promoter in pKOR1. However, many clinical and lab strains of S. The pKOR1 plasmid goes a step further and incorporates a counterselection system based on a tetracycline-inducible transcript that is antisense to the essential secY secretion gene ( 2). ![]() ![]() Moreover, a reoccurring problem with the thermosensitive origins is that their replication is not completely blocked at the nonpermissive temperature, and therefore, if a mutant divides slower than the replication rate of the vector, then it is extremely difficult to isolate plasmid-free cells. Although pMAD encodes a beta-galactosidase gene, which allows blue/white screening for the loss of the vector on plates containing 5-bromo-4-chloro-3-indolyl-β- d-galactopyranoside (X-Gal), in our hands, white colonies could often be shown to retain pMAD, suggesting that the beta-galactosidase is not stably expressed. Other potential problems include temperature sensitivity of the desired mutant and the risk of acquiring secondary mutations during the passages at elevated temperature. This screening can be extremely cumbersome, especially if the desired mutation results in a loss of fitness to the cell, thereby allowing wild-type cells to outgrow the mutant cells during the liquid culture passages. This is usually done by growing the culture under nonselective conditions at 42☌ and by finally screening individual colonies for loss of the plasmid. The next step is to find colonies where the vector has recombined a second time via the other region of homology, thereby losing the plasmid and leaving only the desired mutation. Once colonies have been established after transformation, the temperature is shifted to the nonpermissive temperature of 42☌, while antibiotic selection is kept up, thus selecting for cells where the plasmid has recombined into the chromosome via one of the two regions of homology that correspond to the adjacent regions of the desired mutation. This enables the passage of the vectors through the restriction-defective, but modification-proficient strain RN4220, by maintaining growth at 30☌, before transforming into the final recipient strain ( 14, 25). Common to all of them is that they carry a thermosensitive origin of replication (pE194ts-ORI in pBT2, pMAD, and pKOR1 pT181ts-ORI in pCN39, pCN49, and pCN50), which allows the vectors to replicate in S. aureus via double homologous recombination, including pBT2, pMAD, pKOR1, and a range of pCN vectors ( 1, 2, 5, 7). Several vectors are currently available for generating mutations in S. aureus degradosome as well as the operon containing the cshB DEAD box helicase. Finally, as proof of concept, we present six deletions or modifications of components in the S. The choice between these 12 different pRLY vectors allows for high versatility and ensures that the vectors can be used in virtually any genetic background. The basic vector pRLY2, which contains the pyrFE genes from Bacillus subtilis, was combined with chloramphenicol, erythromycin, and tetracycline resistance genes and four different versions of nonreplicative or conditionally replicative origins of replication. This system allows for stringent counterselection of the vectors during the second homologous recombination of a classic allelic replacement. ![]() The central feature of the vector range is a selection/counterselection system that takes advantage of the 5-fluoroorotic acid (FOA) resistance and pyrimidine prototrophy caused by the loss and gain, respectively, of the pyrF and pyrE genes. We have developed a range of vectors for allelic replacements in Staphylococcus aureus to facilitate genetic work in this opportunistic pathogen.
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