Bb 4 Culture On The Amp Plate example essay topic

Mechanisms of Genetic Exchange: Transformation and Conjugation Introduction Genetic exchange had been known to play a role in the evolution and survival of microorganisms. Genes of one bacterium can be exchanged to another through several different ways: conjugation, transformation and transduction. However, the focus of this paper is to further examine the characteristics of conjugation and transformation mechanisms. Transformation was first discovered in 1928 by Frederick Griffith while he was studying pneumococcal infection in mice. 1 In 1944, Avery, Macleod and McCarthy discovered that the transforming substance is DNA. 1, 2 Transformation occurs when a naked, double-stranded DNA was taken from outside into the cell, may be followed by the integration of the DNA into the host's chromosome, and also replication, transcription and expression of the information encoded in the DNA.

1, 2, 3 However, the recipient cell must be competent- do not secrete DNAase- upon the intake of the DNA fragment (from the donor) in order for the DNA fragments to be recombined into the recipient cell's chromosome. 2 In other words, transformation can be made to be more efficient if the DNAase do not exist since the enzyme acts as a digest or of foreign DNA. 4 An advantage of transformation is the fact that the DNA can be subjected to mutagenesis and other treatments or manipulations, enabling the analysis of the bacterial genome. A disadvantage to the transformation method is the fact that this method can only produce small percentage of transformation in most bacteria. 3 Due to the much smaller sizes of the transforming fragments and their competition for uptake with many other DNA fragments, the probability that a recipient cell incorporated the desired DNA fragment is very slim.

4 Conjugation is another method that is very adaptable and efficient for intra- or inter- species genetic transfer. 3 First discovered in 1946 by Lederberg and Tatum in a strain of E. coli called K-12, conjugation requires physical contact between two genetically different cells via a conjugation tube called the F-plus, which transfers the F factor. 1, 2, 4 The F factor is acquired by conjugation from an F+ to an F-. The F factor has a size of approximately 1/50 the size of the E. coli chromosome and it has no connection to the bacterial chromosome, thus the F factor and the chromosome replicated independently of each other. Eventhough the F factor is not involved in the normal function of the cell and it is dispensable, it is the determinant in the "organism's ability to initiate the conjugal process by which chromosomal DNA, as well as F itself, may be transferred". 1 Donor cell in conjugation is called the "male" (F+) while the recipient cell is called the "female" (F-).

The transfer process of the F factor is also followed by the making of a replicate of the donor's DNA strand via the rolling circle replication. 4 Upon the transfer of the F factor, an F- becomes an F+. If the F factor became integrated into a random location in the bacterial chromosome, the F+ cell becomes and Hfr (High frequency) cell. However, the mating between an Hfr cell with an F- cell rarely resulted in the transfer of the complete chromosome due to the fragility of the DNA thread, thus the F- cell becomes neither an F+ nor an Hfr cell.

2 The recombinant's would be selected for by plating the mating mixture on the media that will only allow the growth of the recombinant with the desired genotype. 4 Materials and Methods Conjugation A line was drawn down the middle for each of the tetracycline (Tet) and ampicillin (Amp) plate, dividing each plate into two sections. One side was labeled BB 4 (F+) and other was labeled SCSI (F-). The appropriate culture (BB 4 or SCSI) was added and spread to the appropriate section. Plates were incubated for 24 hours at 37^0 C followed by analysis and recording of the results.

Approximately 0.1 mL of BB 4 and 0.9 mL of SCSI were added into a test tube already containing 5 mL nutrient broth; incubate for an hour followed by transfer of 0.1 mL into a Tet / Amp plate. The previous procedure was repeated again after four hours of incubation time. Transformation Into each micro centrifuge tube labeled pGREEN and NPC (No Plasmid Control), approximately 50 uL of competent E. coli cells. Also added and mixed into the pGREEN tube was 10 uL of pGREEN, a plasmid containing the gene for Amp resistance and green fluorescent protein which was isolated from Aequorea victoria. However, 10 uL of sterile water was added for the NPC tube. Both tubes were incubated on ice for 45 min, followed by heat shock in 42^0 C heat block for 1 min.

Approximately 0.8 mL of LB broth was added into each of the two-15 mL tubes: pGREEN and NPC. Contents of the two micro centrifuges were transferred into the test tubes accordingly, and were incubated with shaking at 37^0 C for 45 min. Two plates of each LB+Amp and LB (no antibiotic) were obtained and labeled for pGREEN for one of each type, while the other as no plasmid. Into the plates labeled as pGREEN, 0.1 mL of pGREEN culture was transferred and spread.

Similar procedure was done for the no plasmid control culture. Plates were incubated overnight at 37^0 C, followed by analysis and observation of the results. Results Conjugation On the plate labeled for Amp, bacterial growth was observed only in the section labeled for SCSI (F-) E. coli while on the plate labeled for Tet, bacterial growth was observed only in the section labeled for BB 4 (F+). The plate containing the one-hour-incubation mix culture for BB 4 and SCSI was observed to show plenty of bacterial growth, while the plate containing the four-hour-incubation mix culture showed similar result as the one-hour-incubation plate. Transformation The growth of green bacterial colonies can be observed in the LB+amp plate labeled as pGREEN, however for the same LB+amp plate with NPC label, there were no bacterial colonies to be found. For the LB plates labeled pGREEN and NPC, growth of bacterial colonies was apparent, however, none are observed to be green.

Discussion Conjugation The growth of only the E. coli SCSI culture, and not the BB 4 culture, on the Amp plate indicates that the SCSI culture is a strain that is resistant to ampicillin while the BB 4 culture is sensitive to ampicillin. On the contrary, the BB 4 culture growth on the Tet plate indicates that the BB 4 culture is resistant to tetracycline and the SCSI culture is sensitive to tetracycline. The relative culture growths for both the one and four-hour-incubation mix culture of BB 4 and SCSI on Tet / Amp media are very similar, indicating that regardless of the overall period of incubation, the number of bacteria that successfully undergo conjugation is the same. Conjugation is considered successful when the entire F+ plasmid was replicated and the replicate transferred to the previously F- plasmid.

The results gathered from this experiment also showed the rapidity of the conjugation process and its impact on the bacterial community. Relating the speed in which genetic exchange occur in the microbial system to that of human interaction with pathogenic species, one example is a plasmid-mediated drug resistance. Often times called the infectious drug resistance, the plasmid-mediated drug resistance is a case in which a few of plasmid-positive cells converted the entire recipient population into plasmid bearing population in a short time, increasing the resistance to a specific drug. 4 Transformation Results of the transformation experiment correspond to the expected results: the LB+ amp plate containing E. coli transformed with pGREEN yielded green bacterial growth, while the LB+amp plate with no plasmid control (NPC) yielded no bacterial growth. Both of LB containing pGREEN and LB containing NPC produced regular bacterial colonies. The reason why there was no observed bacterial growth for the NPC on the LB+amp plate is the fact that without the pGREEN, the plasmid with gene for green fluorescent protein and ampicillin resistance, the E. coli culture would be sensitive to ampicillin, thus unable to grow on the LB+amp plate.

The addition and uptake of the pGREEN plasmid by some E. coli bacteria enabled them to develop resistance to ampicillin and ability to express the green fluorescent gene. In the LB plates, both the pGREEN and the NPC cultures were able to grow since there was no ampicillin to impede the growth of NPC cultures, and the pGREEN will remain growing with or without ampicillin. Green bacterial growth, however, was not observed for the pGREEN culture contrary to the expectation because...

Bibliography

1. Edited by Glover, S.W., Hopwood, D.A. Genetics As a Tool in Microbiology. Press Syndicate of the University of Cambridge. New York. 1981.
2. Frobisher, M. Fundamentals of Microbiology. W.B. Saunders Company. Pennsylvania. 1968.
3. Burrows, W. Textbook of Microbiology 20th ed. W.B. Saunders Company. Pennsylvania. 1973 4.
Brock, T.D., Madigan, M.T. Biology of Microorganisms 5th edition. Prentice Hall, New Jersey. 1988.