Experiment on Genetic Mapping by Transduction (Essay Sample)

Introduction.
This experiment is based on genetic mapping by transduction. Transduction can be used to determine the distance of genes to each other and therefore to map mutations on the chromosome. Gene mapping involves allocation of loci to a relative position on a chromosome. Gene maps are specific to a species and comprised of genomic markers or genes and the genes distance between each marker. Genetic maps are an important tool for mapping of disease genes or trait loci, in a method known as linkage mapping. This experiment uses transduction in Salmonella enterica to determine the linkage between a transposable element, and the genes encoding tryptophan by synthetic enzymes. Salmonella enterica is a Gram-negative bacteria, motile, non-sporing a facultative anaerobe not a symbiotic but an intracellular pathogen to both human and animals. Salmonella enterica species s subdivided into six sub species: enterica I, salamae II, arizonae IIIa, diarizonae IIIb, houtenae IV and indica VI. Their usual habitat is warm blooded animals and their environments.
All of these spices can infect humans .Salmonella enterica can cause four different clinical manifestations which include gastroenteritis usually characterized by sudden vomiting diarrhea abdominal cramps and nausea, bacteremia which can Cause septic shock in patients over 50yrs, enteric fever, also known as typhoid fever and is characterized by headache, chest rash and fever and an asymptomatic carrier state. Salmonella genome contains a large number of pathogenicity islands synthesizing over 60 effectors.
Some methods of discrimination of both clinical and epidemiological importance include established tests such as phage typing and pulsed field gel electrophoresis. These tests are rapidly being replaced with DNA sequencing.
Salmonella enterica acts as a host I which P22 phage could become established, proliferate and eventually out transduce genetic materials.P22 is able to lysogenize the bacteria and to generate progeny and mediate transduction of a model plasmid contained therein .phage P22 is a bacterial virus that infect salmonella it has an icosahedral head that surrounds its linear double stranded DNA genome and a short tail that binds two the O-antigen of the lipopolysaccharides on the outer membrane. After binding, the phage DNA is translocated across the membranes into the cytoplasm where the P22 genome circulates and initiates several rounds of theta-replication.
The head and the tail proteins are transcribed and translated from the phage genes. The head proteins assemble into empty phage heads and the tail proteins separately assemble into phage tails. After several copies of the phage genome have being produce,P22 switches to rolling circles replication that produces long concatemers of double stranded P22 DNA that are packed into the empty phage heads by headful mechanism. Once the phage head is full, a specific nuclease cuts the DNA left poking out of the phage head. Therefore, the ends of each genome have direct repeats but the ends of the linear DNA is different in each different phage head.
Material and methods used.
Preparation of phage lysate.
This is the first step in the experiment it involves growing a phage lysate on the donor string of bacteria infected with phage P22 and incubated overnight to produce a phage lysate.500 µl of the donor cell culture is added to a tube containing 2ml of LBEDO broth using a P-100 pipetman and labeled carefully. 5 µl of the P22 lysate is added to the donor cell culture and then incubated at 37°C in a vigorously shaking water bath at 16hrs.it is important that one of the lab partners to come back and check the next day in less than 24hrs to remove the phage from the water bath. The tube is gently tilted to check for evidence of lysis of the donor cell and then stored in the 4°C refrigerator until the next step.
Harvesting and tittering phage lysates
This second step involves elimination of any remaining donor cell from the phage lysate. This is done through centrifugation because the phage particles are much smaller than the cells and cell debris, making the cells and the debris to pellets to the bottom of the test tube and the phage will remaining in the supernatant.cloroform is added to the lysate to kill any remaining donor cells that have failed to pellet chloroform works in a way that it disrupts the lipid bi-layer of the cell membranes but the phage capsids which are entirely made of proteins are resistant to chloroform.
1ml of phage lysate is transferred into a fresh micro centrifuge tube using aseptic technique and the remainder is stored in the refrigerator at 4°C. Ensure that the micro centrifuge is balanced and spin for 1 minute at maximum speed. The supernatant, which is the phage, is transferred carefully into a new sterile micro centrifuge tube to avoid the pellet completely. 5 drops of chloroform is then added to the phage lysate tube and gently shaken for 30 seconds to ensure the chloroform fully mixes with the liquid. It is then left to sit for 5 minutes at room temperature and then placed back into the micro centrifuge and spinned for 10 minutes at maximum speed. The supernatant is transferred carefully into a micro centrifuge tube and labeled as “clarified phage lysate” then stored at 4°C.
Transduction.
Considering the phage lysate was grown on a donor string that carries a Tn10 insertion (encoding tetracycline resistant), the recipients strain is tetracycline sensitive and should be infected with the phage lysate at a ratio of one virus to 10 bacteria. The reason for this is; one, to avoid infecting the recipient cell with more than one virus caring bacterial DNA, which could confuse the transduction result and two if the recipient cell is infected with both a transducing particle and an intact phage particle, the intact phage will kill the cell because it will go though its normal replication cycle therefore killing the transductants. The mixture of transducing phage and recipient cell will then be planted on selective media and only the trasdactants that have a resistance to tetracycline will grow on this medium .Those that do not have a resistance will die since the donor cell will not form colonies because they were killed by chloroform during the previous step.
Two controls are important in this transduction experiments. A control with bacteria only (cell control).This is done to ensure that the parental bacteria cannot grow on the selective media, the second control is the one with phage only (phage control) done to ensure that the phage lysate is not contaminated with bacteria that can grow in selective media
Plaques on each titer plate is counted and the titer of the phage lysate is determined using the appropriate dilution factor. 100 µl of receipts cells is used to make an appropriate dilution of the phage lysate together with 100 µl of the dilution to the cells. One of the plate is labeled LB+Tet plate. 100 µl of the recipient bacteria and 100 µl of the diluted phage lysate is thoroughly and evenly spread