In this lab we observed the growth of bacteria in a conjugation and transformation experiment. We expected the mating plate of the conjunction to grow and the bacteria on the transformation plates to grow also. We also practiced using aseptic techniques to clean the lab benches and to keep the bacteria from being contaminated from outside bacteria. This lab will take a total of 2 days because of the observations that need to be made. The parts of this paper will explain the procedure and the observations of the bacteria plates.
This lab was presented in the General Biology 103 Lab Manual by the Department of Biology, Fall 1999 edition on pages 106-118. In this lab we first used specific aseptic techniques to properly transfer bacteria from test tubes to petri dishes. The lab bench was cleaned with a 10% Clorox bleach solution and left to dry while my lab partners and myself washed our hands. A Bunsen burner was provided so the inoculating loop and the neck of the test tubes could be placed in the flame to kill off any bacteria that was on it. After the inoculating loop and test tube neck are cooled the loop is placed into the tube and then swabbed across the surface of the agar plate. The lid of the plate is not totally removed so airborne organisms cannot infect the culture being placed on the dish. This procedure must be followed every time a new strain of bacteria is placed on the dish. After the aseptic techniques are followed four petri dishes are obtained and labeled with the type of bacteria that will be placed in it. After the strains of bacteria have been placed in the petri dish the plates must be incubated at 37C and left for the next day. Finally a mating plate is made by letting the two strains touch inside the petri dish and then be placed in a 37 incubator for one day.
For the next part of the lab transformation will be studied. This procedure follows the procedure for conjugation in the lab manual. First 10ul of pBLU plasmid with the lacZ gene and ampicillin resistance will be added to 250ul of E. coli cells and left on ice for 20 minutes. Next 10ul of nutrient broth will be added to 250ul of E. coli and placed on ice for 20 minutes. The nutrient broth is a combination of sugars, minerals and amino acids. After the 20 minutes is up the tubes will be placed in a 42C water bath for 90 seconds. The temperature shock aids the E. coli in transforming with the plasmid. After the 90 seconds the tubes are put back on ice. Next 0.5ml of nutrient broth are added to both tubes and placed in a 37C water bath for 20 minutes. After 20 minutes 3 agar plates are obtained and labeled and the contents of the tubes are placed on the plates. The plates are then left for 2 days and the results will be recorded. ON the second day the observations for the conjugation are made and then the plates are discarded except for the mating plate. Three new plates are obtained and labeled and the cells from the mating plate are placed in each of the three new dishes. These dishes are placed in the incubator and observed the next day along with the transformation dishes.
After one day the plates that were placed into the incubator were retrieved and observations were made. All of the conjugation plates had streptomycin on one side and ampicillin on the other. In the first plate containing LB agar only we observed that the bacteria grew for both the streptomycin and ampicillin. The plate with the LB and the streptomycin produced growth on the streptomycin but the ampicillin had no growth. The next plate with the LB and ampicillin produced growth on the ampicillin but no growth on the streptomycin side. Finally the plate with LB, streptomycin, and ampicillin produced no growth on both sides of the dish. The results table with this information is attached to this report and labeled as 1-1.
After two days the transformation dishes were removed from the incubator and examined. The dish with the LB agar only produced growth on both sides. The dish with LB and ampicillin produced growth on both sides and finally the LB, ampicillin and Xgal produced growth on both sides. The results table for these observations are included at the end of this report and labeled 1-2.
Finally the last plate was the mating plate for the conjugation. The plate with the LB and Ampicillin produced growth. The plate with LB and Streptomycin produced growth and the plate with LB, ampicillin, and streptomycin produced growth. The table for these observations is 1-3.
All of the observed results presented in the graphs are positive results that matched the results we expected to get. These results prove that our bacteria went through conjunction and transformation.
In the conjunction experiment the starting genomes were streptomycin and ampicillin. Both of these strains were antibiotics and produced growth on the plates where the same bacteria strain was present. These strains produced a third strain that is resistant to both ampicillin and streptomycin. Through our observations we were able to prove conjugation took place because there was growth on the LB Strep, LB Amp, and LB, Strep, Amp plates. Because of the growth on all three plates I came to the conclusion that the DNA was shared between both strains of bacteria. If we look at table 1-1 we can see that there was no growth for either streptomycin or ampicillin in the plate with LB, strep, and amp. According to the results recorded in table 1-3 we observed growth for the plate that had LB and both streptomycin and ampicillin on it. I came to the conclusion that the conjunction was successful because of the growth on the mating plate. If the conjunction did not take place I would have expected the same results that were observed in table 1-1. Growth on the mating plate proved to be a success because we were looking for growth and we got it. It was also good to use streptomycin and ampicillin because they are antibiotics and they prevented any other strain of outside bacteria from growing on the plates.
Conjunction of bacteria can be very useful in the medical world. If we have two types of bacteria and they are placed into a plate and allowed to physically touch we would be able to make bacteria to fight off many types of diseases. This experiment proved that placing two antibiotics on the same plate will create a new strain of bacteria with both strains DNA present and not let any outside bacteria get in to ruin the results. Conjunction can be used to kill bacteria with resistance genes to many common bacteria. An antibiotic with two sets of DNA will kill the donor strains and any other strains the donor shared its plasmid with.
For the transformation experiment the two genomes used were pBLU and nutrient broth. Xgal is also used in this part of the experiment. The Xgal is a synthetic molecule which is almost molecularly identical to lactose. The Xgal is so identical to lactose that beta-galactosidase will recognize Xgal as lactose. The beta-galactosidase cleaves the Xgal and produces glucose and a blue precipitate. Bacteria cells with the lacZ gene for beta-galactosidase grow on the plate with the Xgal and turn a blue color. The blue color indicates that the beta-galactosidase is working and protein is being made on the bacteria plasmid.
In the process of transformation there is an uptake of exogenous DNA to create a genetic trait that is stable and heritable. During this process the recipient bacteria cells must be in a particular state before they can be transformed. The state before transformation is called competency. Competency occurs naturally in species but can also be done experimentally. The cells must be incubated on ice for 12-24 hours in a calcium chloride solution. The solution punches holes in the cells so the plasmid can enter. Competency is extremely necessary for a successful transformation.
These cells were also heat shocked before they were incubated and left to be observed for the next two days. Heat shock is when the cells are suddenly exposed to extreme temperatures. The cells were left on ice and then placed into a 49C for 90 seconds and then placed back on ice. The sudden change in temperature induces the bacteria to uptake foreign DNA into the cytoplasm through the holes created by the CaCl. The temperature change creates a draft to carry the DNA into the cell. The DNA becomes part of the chromosome as and exists as an extra chromosomal circular plasmid. The number of cells on the plate must be very high in number because there is only a 10-30% chance of recombinant cells from the original batch of cells.
In this lab I concluded that my bacteria went through conjugation and transformation because of the growth on the plates.The growth is a positive reaction for this experiment.