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Lab Report

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Biology
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Wayne Community College
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Lab report
Introduction:
The experiment(Lab.Manual Exp.7) is done to understand genetic mapping through
transduction. The experiment was done by using Bacteriophage p22 transduction in salmonella
enterica_serovar Typhimurium. The purpose of the experiment is to determine and understand
the linkage between transposable elements (Tn10 tetracycline-resistant sequence, and the
tryptophan (Trp) operon).one of the three methods for horizontal gene transfer is used in this
experiment, in which a virus is infecting the bacterial cell, proliferate and multiply its genome
and lysis leads to infection of other cells. the lysis is done by lysate protein after forming 50-
100 copies inside the host(Lab.Manual Exp.7 page83-84). Phage p22 has an O-antigen on its
head which encapsulates its double-stranded DNA. This leads to attachment and transfers its
genome to the host and makes new copies. These new copies are released through a process
termed cell lysis. Now the newly synthesized copies infect other cells in culture. The lysed
cells are removed by the process of centrifugation and unlysed cells are killed by using
chloroform. The solution containing the lysed cells can be stored in chloroform. This process
is termed general transduction(Lab.Manual Exp.7 page82) in which the pac region on the virus
genome is present which is the initiation site for replication. such sequence also present on host
genome leads nuclease to cut host genome instead of the viral genome. the occurrence of a
similar sequence on the host organism is due to the high probability of finding a short sequence
on a large polynucleotide because the sequence is made up of only 4 nucleotides a sequence
repeats itself after every 4 nucleotides. the nucleotide sequence of the virus replicates in a
rolling circle fashion and gets accumulated inside the head of size about 44kbp (kilobase pairs).
To counter this a modified p22 with high transducing(HT) frequency is used which does not
cut the identical sequence to pac as well as it contains a Tn10 and trp. Therefore the donor
contains tetracycline-resistant genes while the host does not have such resistance. such resistant
phenotype is considered as selective markers. Only those recombinant cells that have
tetracycline resistance will be able to grow in a media enriched with the antibiotic. Trp
+
or
prototrophs are considered those cells which able to grow without tryptophan while auxotrophs
(trp
-
) (Lab.Manual Exp.7 page86)not be able to grow without tryptophan. Using tetracycline
resistance is considered as the selection of the recombinants while screening is done using the
Tryptophan.
Materials and methods:

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This experiment is performed in various labs. These labs are performed in a stepwise manner
to obtain results.
The first lab(Lab.Manual Exp.7 page88) is to prepare the lysate and incubation the lysate
obtained. For that sterilized tubes and pipetman are required. LBEDO broth is used to grow
donor cells Salmonella enterica Trp
+
:: Tn10 and phage p22 HT and incubated for 24 hours at
37
o
C. After 24 hours these cells' lysate is kept at 4
o
C. the overnight incubation leads to proper
growth and formation of lysate.
The second lab(Lab.Manual Exp.7 page89) involves the harvesting of lysate and titrating. After
the first step, the cell lysate is well prepared and now these need to be harvested. Materials
required for these steps are pipetman and sterile tips, microcentrifuge, sterilized
microcentrifuge tubes, and chloroform(CHCl3). After lysis, phages are suspended and these
are very small in size compared to the cell debris and other cells. Using a micropipette the
lysate is prepared and stored after the first step is poured in centrifuge tubes. Centrifuge for 1-
minute. Phage due to small size and density remains in the upper clear layer while cell debris
is at the bottom. Carefully remove the clear part and transfer it to a fresh pipet. Add some
drops of CHCl3 and vortex it for 5 minutes. The remaining cell’s lipid layer is disrupted by
chloroform and then centrifuge for 10 minutes. Balance the centrifuge by placing other tubes
on opposite ends. All phages are in a clear layer and then carefully transfer this clear layer of
lysate in a fresh tube and stored at 4
o
C. the phages are resistant against chloroform because
the capsid of the phage is made up of proteins.
Titrating the lysate(Lab.Manual Exp.7 page90): the lysate obtained from the previous step is
used to count the pfu/ml. this can be done by plaque assay. The step requires sterilized pipet
tips, water bath at 50
o
C for keeping the melted agar. Firstly prepare 8 dilutions of phage lysate
with a buffer from dilution 10
-1
to 10
-8
. The plates should be kept in the compartment where
sterile conditions are maintained until used for the experiment. Quickly transfer the agar on
two plates and let them solidify. When it gets solidify pour a small amount of each diluted
lysate 4 on each plate and mark them with their dilutions(Lab.Manual Exp.7 page91). The
lysate infecting the first cell I then transfer ed to neighboring cells and make a clearing of cells,
this is known as a plaque. These plates are incubated at 37 C overnight and the next day pfu/ml
should be recorded.
The multiplicity of infection (MOI) (Lab.Manual Exp.7 page93) Is the number of a cell infected
by the lysate divided by the total number of cells. If the MOI of any sample is 1 then it is

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Lab report Introduction: The experiment(Lab.Manual Exp.7) is done to understand genetic mapping through transduction. The experiment was done by using Bacteriophage p22 transduction in salmonella enterica_serovar Typhimurium. The purpose of the experiment is to determine and understand the linkage between transposable elements (Tn10 tetracycline-resistant sequence, and the tryptophan (Trp) operon).one of the three methods for horizontal gene transfer is used in this experiment, in which a virus is infecting the bacterial cell, proliferate and multiply its genome and lysis leads to infection of other cells. the lysis is done by lysate protein after forming 50100 copies inside the host(Lab.Manual Exp.7 page83-84). Phage p22 has an O-antigen on its head which encapsulates its double-stranded DNA. This leads to attachment and transfers its genome to the host and makes new copies. These new copies are released through a process termed cell lysis. Now the newly synthesized copies infect other cells in culture. The lysed cells are removed by the process of centrifugation and unlysed cells are killed by using chloroform. The solution containing the lysed cells can be stored in chloroform. This process is termed general transduction(Lab.Manual Exp.7 page82) in which the pac region on the virus genome is present which is the initiation site for replication. such sequence also present on host genome leads nuclease to cut host genome instead of the viral genome. the occurrence of a simil ...
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