Antibiotic resistance markers, plasmid

Figure 4, Construction of plasmids harboring mobile expression elements containing structural genes for the conversion of toluene to p-cresol, and p-cresol to HBA. (a) pESMll and (b) pESM23. The structure of the mini-TnS transposons is emphasized in the figure for clarity. The DNA sequences coding antibiotic resistance markers aphA and tet, regulatory proteins lacfi and nahR, and promoters Ptrc and Psal are indicated. The mobile units are present in the delivery plasmid pUT as XbaTEcoRI restriction fragments (44).

Recent achievements in molecular biology have created multiple methods that can be utilized to construct mutants by allelic replacements in different bacteria. A variety of temperature-sensitive plasmids, antibiotics resistance markers, and transformation protocols are now available. 

Add SOC media with no antibiotic to a final volume of 60-100 pL/tube. For plasmids using the ampicillin resistance marker, the cells will begin repairing their cell walls immediately and are ready to be plated. For plasmids with other antibiotic resistance markers, incubate without shaking for 20 min before plating. 

To identify bacteria which have taken up the vector, a common ploy is to utilize a characteristic of the vector, i.e. a vectorial marker. Plasmids possessing an antibiotic-resistance marker will enable a microorganism to grow in culture medium containing that antibiotic. Bacteria lacking the plasmid fail to develop colonies. For example, the plasmid pBR 322 contains antibiotic-resistance markers for tetracycline and ampicillin. Therefore, bacteria transformed by this plasmid are capable of colony formation in tetracycline- and/or ampi-cillin-containing medium and are easily selected. 

Yeast-bacteria shuttle plasmids are usually able to be maintained in both E. coli and yeast and can be episomal or integrated into the host genome. First, plasmid DNA is usually amplified in E. coli before yeast gets transformed. Different antibiotic resistance cassettes are available, and also an abundance of autotrophic markers was established. 

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