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. oli 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 transductants were tested by patching them onto a Petrie plate lacking tryptophan and uracil, which will allow growth of trp+, pyrF+, and not the trp+,pyrF- transductants. Trp gene encodes for trypsin, while the pyrF gene encodes for pyramidines, such as uracil.
Thi, his, and pro genes encode for thiamine, histidine, and proline respeictively (1). Finally, the frequency of transductant was used to obtain the distance between the trp and pyrF gene. Discussion In the transformation experiment, E. coli cells were transformed with Plasmid DNA containing the tetracycline resistant gene, and were tested for competency by growing them in LB in presence of tetracycline antibiotic. Tetracycline is a broad spectrum antibiotic that works by inhibiting translation, thus inhibiting protein synthesis.
It attaches to the 30S subunit of ribosome and prevents the charged aminoacyl-tRNA from binding (3). As shown on Table 1, Tube 3 and Tube 2 did not have any growth of E. coli cells. Tube 2 contained E. coli cells only, and when cultured on the LB plate in presence of tetracycline antibiotic, the growth was inhibited by the antibiotic. Because the E. coli cells in tube 2 lacked the tetracycline resistant gene contained in the plasmid DNA, it was unable to grow in the presence of tetracycline antibiotic. Tube 3, lacked the E. coli cells and so, there were no cells to grow in the LB plate with or without the tetracycline.
Tube 3 instead was used as a control, to test for contamination in the plasmid DNA and the calcium chloride. In tube 1, which contained the E. coli cells and the plasmid DNA containing the tetracycline resistant gene, transformation occurred. As a result, E. coli cells have acquired the tetracycline resistant gene, being able to grow on the LB plate in the presence of the tetracycline antibiotic. Therefore the viable competent cells were counted from tube 1 contents in 102 diluted LB (with antibiotic), which had cells between 30 and 300 colonies.
On the other hand, the total viable cells were counted from tube 1 in 106 diluted plate that was grown on LB without the antibiotic. As shown on table. 2, the viable competent cells were calculated to be 24350 cells/100ul and the total viable cells were found to be 370,500,000 cells/100ul. Finally, the transformation frequency, which is the ratio of transformants per viable cell, was calculated and was found to be 6. 49 x 10-05 as shown on table 2. In the generalized transduction experiment, trp gene from the donor strain of CSH61 (P1vir lysate) was laterally transferred to the recipient strain, CSH54.
CSH61 strain are trp+, pyrF+ and the CSH54 strain are trp-, pyrF-, and because trp and pyrF are linked together on the same chromosomal fragment, they are cotransduced (1). The transductants were identified by selecting for the trp+ marker by growing in absence of tryptophan, however the pyrF marker may be present or absent, depending on the crossover event during recombination. This was identified by patching the transductants onto a Petrie plate lacking tryptophan and uracil. The trp+, pyrF+ transductants will be able to synthesize both tryptophan and uracil, and therefore will grow in this minimal medium (1).
However, trp+, pyrF- transductants cannot grow in the medium, because they won’t be able to synthesize uracil (1). As shown on table 3, 57. 5% of transductants were trp+, pyrF+ while 42. 5% were trp+, pyrF-. This means trp is cotransduced with pyrF at 57. 5% frequency, which indicates that they are very closely linked. Markers which are separated by less than 0. 5 minutes are cotransduced at 35-95%, and so, trp which cotransduced with pyrF at 57. 5%, is very closely linked (1). This closely matches the actual cotransduction frequency of 55% between trp and pyrF in E. oli (2). According to the Wu formula, the cotransduction frequency of 57. 5% yielded distance of 0. 084 minutes, which was very close to the distance of 0. 09 minutes at cotransduction frequency of 55% found in literature (2). Bacterial genes can also be analyzed by a method known as Southern blotting (4). In this method, DNA is treated with restriction enzymes, which cuts the DNA into fragments of different size. Then the fragments are run on an agarose gel by electrophoresis, which separates the fragments by size.