WebNov 6, 2013 · When Tet/SacB counter-selection agar is used during a P1 transduction , it is modified by adding 5 ml of a sterile 1 M sodium citrate solution to the agar for a final concentration of 5 mM. This is the P1 Tet/SacB counter-selection agar. Creating the tetA-sacB counter-selection cassette. WebI have a problem getting my sacB counter-selection to work. sacB sequence (obtained from PYUB870) was cloned into the MCS of pGEM-T, transformed into E. coli and plated on 1) 5% sucrose LB agar ...
Harnessing Bacterial Toxins for Allelic Exchange - Addgene
WebOct 1, 2024 · A knockout vector (pMTL70621-SacB) was also constructed carrying the sacB counter selection marker. Through provision of the appropriate mutant alleles, the vector was subsequently used to delete genes encoding restriction enzymes by selecting cells able to grow in the presence of 15% ( w / v) sucrose (see Supplementary Information). WebMar 20, 2024 · Seamless modification of the Escherichia coli genome using positive selection / negative selection is widely used in metabolic engineering and functional genome analysis. Some excellent negative selection systems have been reported, of which tetA-sacB and inducible toxins system are prominent. To expand the existing negative … primary cover gasket sportster
Expression of the Bacillus subtilis sacB gene confers sucrose ...
WebMar 20, 2024 · The selection stringency of kil was measured and compared with the most widely used counter-selection gene, sacB, at the lacI, ack, and dbpa loci using different E. coli strains. At all these loci of tested strains, the selection stringency of kil significantly exceeds that of sacB by 2- to 28-fold. When dsDNA fragments were employed for ... WebMar 22, 2016 · Sucrose-LB agar (5% concentration) was used for sacB gene-based counter selection in mutant strains’ construction. 4.2. Construction of Plasmids and Mutations. The primers designed in this study are listed in Table S1. The protocol of DNA manipulations was performed as previous described . WebNov 19, 2014 · The cat-sacB cassette facilitated generation of multiple successive mutations in A. pleuropneumoniae using the two-step transformation protocol. Cm selection was very stringent, and all Cm-resistant clones tested were confirmed to contain the cat-sacB cassette by PCR (data not shown). play disney sorcerer\u0027s