Transformation Study Materials

Antibiotic Function and Resistance Genes Antibiotic Function Ampicillin is classified as a beta-lactam antibiotic. It is in this family based on its structure; it contains a beta-lactam ring Ampicillin acts as a competitive inhibitor for the transpeptidase enzyme which is involved in cell wall synthesis, specifically in forming the peptidoglycan layer. Interfering with cell wall formation leads to a variety of problems for bacteria, and death usually occurs via lysis Kanamycin is another common antibiotic that is classified as an aminoglycoside antibiotic. It inhibits protein translation by targeting bacterial ribosomes Kanamycin binds to the 165S rRNA component of the 30S subunit of the bacterial ribosome. It induces a change in the conformation of the complex formed between the mRNA codon and the tRNA. This promotes tRNA mismatching resulting in protein mistranslation Antibiotic Resistance Genes The ampicillin resistance gene encodes an enzyme called beta-lactamase. It cleaves the beta-lactam ring in ampicillin, rendering the antibiotic nonfunctional. Beta-lactamase is classified as an antibiotic degrading enzyme The kanamycin resistance gene encodes a kinase called aminoglycoside kinase. The kinase phosphorylates kanamycin, modifying the antibiotic so it can no longer interact with its target. Aminoglycoside kinase is classified as an antibiotic modifying enzyme Competent Cells and Transformation Introduction to bacterial transformation Bacterial transformation was first observed in 1928, but it was not until 1944 that Avery, McLeod, and McCarthy performed the first definitive experiment indicating that the "transforming principle" was really DNA. However, a large number of bacteria (such as the lab strains of E. coli) are not naturally competent and must be treated with chemicals and altered temperature in order to induce competence Competency and Transformation Competency is defined as the ability of a recipient cell to take up foreign DNA. Bacterial transformation is defined as the actual physical process of taking the DNA up from the environment or other cells The steps involved in this process are: (1) DNA uptake (2) Cytoplasmic transport and modification (3) Possible recombination with the recipient chromosome (unless it is plasmid DNA) (4) The expression of the transformed gene(s) An important advance in molecular biology was the use of extrachromosomal DNA molecules, plasmids, to carry transforming genes Plasmids are independently replicating pieces of DNA, and so do not need to undergo the recombination step to transform a cell and have its genes expressed Limitations of E. coli cells E. coli are not naturally competent (i.e. Does not naturally take up foreign DNA on its own) and therefore is difficult to transform with foreign DNA. However, it can be made artificially competent by exposing it to high calcium concentration and transformed using heat shock It is also important to realize that when E. coli are transformed, usually only circular DNA molecules are successfully transformed. Linear DNA is rapidly destroyed by the E. coli Serial Dilutions Why do we need serial dilutions? Serial dilutions are a series of dilutions where one dilution is used to make another dilution that is then used to make another dilution Single dilution factors Single dilution factors tell you the amount that you diluted each dilution. To calculate single dilution factors, divide final volume by volume transferred Overall dilution factors Overall dilution factors tell you the difference between each dilution and the stock culture. To calculate the overall dilution factors, multiply the dilution factors for each single dilution Determining the concentration of the stock and total cells We assume each bacterial colony originates from only 1 bacterial cell, so we assume 1 colony is equal to 1 cell. To determine Cell density (cells/ml) of the dilution take the # of colonies on a plate divided by the volume plated for that dilution If A concentration is known for a diluted sample, then the concentration of the original stock can be determined by multiplying the diluted concentration by the overall dilution factor for that dilution