The First
In the first part of this lab, E.coli cells were transformed with an R-plasmid carrying a tetracycline resistant gene, giving rise to tetracycline resistant E.coli strain. This was accomplished through transformation, which allowed E.coli to directly uptake the naked DNA molecule carrying the antibiotic resistant gene (1). However, in order to take up the DNA and incorporate them into their genome via recombination, cells must be competent (1). Therefore, E.coli cells which are not competent under normal conditions were treated with cold and high concentration of CaCl2, in order to make them artificially competent (1). The transformants were grown on the LB with the tetracycline antibiotic, and on the LB without the tetracycline. Then the viable competent cells and the viable cells were counted to calculate the frequency of transformation.
In the second part of the lab, lateral gene transfer by generalized transduction was done on E.coli cells. In the process of transduction, the transfer of genes is facilitated by bacteriophage, which is a virus that infects a bacterial host (1). Generalized transduction involves lytic infections that kill the bacterial cells, and during the process, bacterial DNA is packaged into a new phage head which in turn injects the DNA into another bacterium (1). In this lab, P1vir phage was used and grown on the donor strain by making a phage lysate. P1vir phage kills bacterial cells by lytic infections, which is required in the generalized transduction (1). On the other hand, the wild-type p1 is a lysogenic phage and therefore could not be used for the generalized transduction (1). In order to prevent excessive killing of the recipient E.coli strain, the P1vir lysate was tittered by serial dilutions. This would also prevent infection and lysis of the transducing particle. In generalized transduction, trp-pyrF region of CSH61 chromosome, which was the P1vir lysate, was laterally transferred to the recipient CSH54 strain. The genotypes of
In the second part of the lab, lateral gene transfer by generalized transduction was done on E.coli cells. In the process of transduction, the transfer of genes is facilitated by bacteriophage, which is a virus that infects a bacterial host (1). Generalized transduction involves lytic infections that kill the bacterial cells, and during the process, bacterial DNA is packaged into a new phage head which in turn injects the DNA into another bacterium (1). In this lab, P1vir phage was used and grown on the donor strain by making a phage lysate. P1vir phage kills bacterial cells by lytic infections, which is required in the generalized transduction (1). On the other hand, the wild-type p1 is a lysogenic phage and therefore could not be used for the generalized transduction (1). In order to prevent excessive killing of the recipient E.coli strain, the P1vir lysate was tittered by serial dilutions. This would also prevent infection and lysis of the transducing particle. In generalized transduction, trp-pyrF region of CSH61 chromosome, which was the P1vir lysate, was laterally transferred to the recipient CSH54 strain. The genotypes of