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 pl

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.

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.

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.

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 49°C 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.

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.

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Approximate Pages = 6 (250 words per page double spaced)