Bacterial Conjugation Experiment
INTRODUCTION: Bacteria, in general, reproduce asexually, but in order to increase diversity, they have developed a mechanism for transfer of genetic material from one bacterium to another. The ability to perform this transfer is conferred by a set of genes which are called F for 'fertility.' These genes exist on a small, circular piece of deoxyribonucleic acid (DNA) that replicates independently from the bacterial chromosome, or they can be integrated into the chromosome. The bacterium containing this gene (sometimes referred to as 'male' or F+) extends its pilus to a neighboring bacterium (sometimes referred to as 'female' or F-), and the two cells are attached. This process is called conjugation. The third manifestation of the F factor is Hfr, which is the term for the F element becoming integrated into the genome. When conjugation occurs, the F genes start travelling across the pilus, bringing the remainder of the genome behind it. Most often, the entire genome isn't transferred. The bacterial genome that is delivered can be measured in minutes from the origin of transfer. That is, the amount of time it takes for a particular gene to be transferred from one bacterium to another indicated how far it is from the origin of replication.
METHODS AND MATERIALS: Media Preparation: The starting material was Medium 56-glucose agar (MM560). The components of the MM56 are found in figure 1. From this, we made two types of media, complete and selective. The reagents used, along with their stock and final concentrations are found in figure 2. The formula:
Stock volume = [(Final concentration)(final volume)] / (stock concentration)
was used to calculate the amounts of each reagent added to th...
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Proline deficient plates
Time 10-1 10-2 10-3 10-4
0 minutes NG NG NG 3
10 minutes 37 NG TNTC 44
20 minutes 49 TNTC TNTC 58
30 minutes 43 TNTC TNTC 40
40 minutes 11 5 TNTC 94
50 minutes 53 TNTC 50 51
60 minutes 14 NG TNTC NG
Histadine deficient plates
Time 10-1 10-2 10-3 10-4
0 minutes 10 2 NG NG
10 minutes 46 43 26 3
20 minutes TNTC 136 NG 4
30 minutes TNTC 184 8 3
40 minutes TNTC 340 20 NG
50 minutes 45 TNTC 27 3
60 minutes 41 250 42 NG
TNTC = Too numerous to count
NG = No growth
Figure 4 - Time of entry table (percentages of recombinants)
Time Thr - Pro - His -
0 minutes .44 0 0
10 minutes 5.8 2 .02
20 minutes 6.7 2.6 .06
30 minutes .004 1.8 .08
40 minutes .0098 4.3 .09
50 minutes 1.9 2.3 .002
60 minutes 2.4 0 .11
Figure 5 - Time of entry graph (percentages of recombinants)
Figure 6 - Map of E. coli K12
pBK-CMV is a plasmid vector 4518 in size, it also contains a multiple coding site (polylinker) that has recognition sequences for many restriction endonucleases. cDNA molecule CHI-1, which is 600bp, has been previously inserted. pUC19 is a cloning vector developed by….. in …….at….(REF). This vector is 2686bp in size and contains a 54 base pair (bp) polylinker containing 13 specific restriction sites, Xba1 and EcoR1 inclusive. It makes a good cloning vector as it is small in size, this makes it easier to be taken up by its host during transformation and allows for a faster replication time (Green, 2015). It contains an origin of replication pMB1 which is essential to be able to replicate. pMB1 has a high copy number allowing for multiple copies to be made (REF hcn pmb1). The pUC19 plasmid vector contains an ampicillin resistance gene, the host containing this plasmid will survive in the presence of ampicillin allowing for the selection of transformed host bacteria. The polylinker of pUC19 is contained within a lacz’ gene allowing us to distinguish between recombinant pUC19 and non-recombinant pUC19 through a process call insertional inactivation (Green, 2015).
It has an outer membrane that contains lipopolysaccharides, a periplasmic space with a peptidoglycan layer, and an inner cytoplasmic membrane. It also consists of adhesive fimbriae. Some strains of E. coli are piliated and are capable of accepting, as well as transferring plasmid to and from other bacteria. This enables the bacteria under stressful or bad conditions to survive. Although its structure is simple with only one chromosomal DNA and a plasmid, it can perform complicated metabolism to help maintain its cell division and cell growth. E. coli produce very rapidly; a single microscopic cell can divide to form a visible colony with millions of cells overnight (phschool.com). It is the preferred bacteria in most laboratories because it grows fast and easy, and can obtain energy from a wide variety of sources. Since the birth of molecular cloning, E. coli has been used as a host for introduced DNA sequences (biotechlearn.org.nz). In 1973, Boyer and Cohen showed that two short pieces of DNA could be cut and pasted together, and returned to
The ingredient of the production need is magnesium sulfate and potassium phosphate at the concentrations of 0.4 and 1.0 g per liter. The concentrations of Fe and Zn is needed at 0.25ug/ml of Fe and 0.575 μg/ml of Zn, respectively, whereas Mo at 0.04 μg/ml allows maximium cellular growth and antibiotic synthesis.
The purpose of this experiment is to identify an unknown insert DNA by using plasmid DNA as a vector to duplicate the unknown insert DNA. The bacteria will then be transformed by having it take in the plasmid DNA, which will allow us to identify our unknown insert as either the cat gene or the kan gene.
Materials used in the experiment included 5-7 g of the potato tissue, 50ml of 2.0M phosphate buffer coffee filter and guaiacol dye.
The actual amount of crude product was determined to be 3.11 grams. The percent yield of the crude product was determined to be 67.75 %. The actual amount of pure product formed was found to be 4.38 grams. The percent yield of the pure product was determined to be 95.42%. Regarding the thin layer chromatography, the line from the solvent front is 8 centimeters.
23. Official methods of analysis, AOAC, Association of Official Analytical Chemists. Edition 17: AOAC, 2003.
This diploid zygote, which undergoes meiosis, forms eight haploid ascospores. The ability of Sordaria to make 8 haploid ascospores is what makes it unique and important for the laboratory exercise done in the lab. The laboratory performed had as a purpose to determine the change in crossing-over frequency or the also called map distance under different environmental conditions in Sordaria sp. This paper has a purpose to analyze, with the help of scientific research, the results obtained in the laboratory exercise. To investigate the crossing over in Sordaria sp., a cross between two mutant strains was performed.
Antibiotic resistance is bacteria’s loss of susceptibility to the bactericidal or growth-inhibiting properties of an antibiotics. When a resistant strain of bacteria is the dominant strain in an infection, the infection may be untreatable and deadly he primary mechanisms of bacterial gene transfer are transduction and conjugation. Transduction occurs when a bacterial virus, called a bacteriophage, detaches from one bacterial cell, carrying with it some of that bacterium’s genome, and then infects another cell. When the bacteriophage inserts its genetic content into the genome of the next bacterium, the previous bacterium’s DNA also is incorporated into the genome. Conjugation occurs when two bacteria come into physical contact with each other and a plasmid, sometimes carry...
On the other hand, cells that have resistance from the start or acquire it later may survive. At the same time, when antibiotics attack disease-causing bacteria, they also attack benign bacteria. This process eliminates drug-susceptible bacteria and favors bacteria that are resistant. Two things happen, populations of non-resistant and harmless bacteria are diminished, and because of the reduction of competition from these harmless and/or susceptible bacteria, resistant forms of disease-causing bacteria proliferate. As the resistant forms of the bacteria proliferate, there is more opportunity for genetic or chromosomal mutation (spontaneous DNA mutation (1)) or transformation, that comes about either through a form of microbial sex (1) or through the transference of plasmids, small circles of DNA (1), which allow bacteria to interchange genes with ease.
== § Test tubes X 11 § 0.10 molar dm -3 Copper (II) Sulphate solution § distilled water § egg albumen from 3 eggs. § Syringe X 12 § colorimeter § tripod § 100ml beaker § Bunsen burner § test tube holder § safety glasses § gloves § test tube pen § test tube method = == = =
The birth of genetic engineering and recombinant DNA began in Stanford University, in the year 1970 (Hein). Biochemistry and medicine researchers were pursuing separate research pathways, yet these pathways converged to form what is now known as biotechnology (Hein). The biochemistry department was, at the time, focusing on an animal virus, and found a method of slicing DNA so cleanly that it would reform and go on to infect other cells. (Hein) The medical department focused on bacteria and developed a microscopic molecular messenger, that could not only carry a foreign “blueprint”, or message, but could also get the bacteria to read and copy the information. (Hein) One concept is needed to understand what happened at Stanford: how a bacterial “factory” turns “on” or “off”. (Hein) When a cell is dividing or producing a protein, it uses promoters (“on switches”) to start the process and terminators (“off switches”) to stop the process. (Hein) To form proteins, promoters and terminators are used to tell where the protein begins and where it ends. (Hein) In 1972 Herbert Boyer, a biochemist, provided Stanford with a bacterial enzyme called Eco R1. (Hein) This enzyme is used by bacteria to defend themselves against bacteriophages, or bacterial viruses. (Hein) The biochemistry department used this enzyme as a “molecular scalpel”, to cut a monkey virus called SV40. (Hein) What the Stanford researchers observed was that, when they did this, the virus reformed at the cleaved site in a circular manner. It later went on to infect other cells as if nothing had happened. (Hein) This proved that EcoR1 could cut the bonding sites on two different DNA strands, which could be combined using the “sticky ends” at the sites. (Hein). The contribution towards genetic engineering from the biochemistry department was the observations of EcoR1’s cleavage of
Materials and Methods: An ion exchange chromatography column was obtained and set up for purification with the addition of 0.5 ml ion exchange matrix. 1 ml
Prepare .05 to .075 g of crude lipid by dissolving it in hexane. Add to the silica gel slurry in the column.
Knowledge is power when discussing the classes you have taken in college and how it affects your everyday life. Microbiology is one of those important classes where although for a majority of majors you do not have to take it and therefore will not learn the information discussed in it, other majors such as biology and nursing require you to take it. They will require you to take it because you will encounter some of the information being taught in it in the work field. As a nurse practitioner I have worked in the field with many patients who were diagnosed with Leprosy, Escherichia coli, Salmonella and other bacterial diseases. All of these diseases that I have encountered in patients were ones I was familiar with due to learning about them as a student in microbiology at CSUB. My children plan on