take homologous recombination) donor strain into a recombination proficient

take out questions

BI324

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Genetics and Evolution

Lab report of the transduction practical

Mon: 22.01.2018 (noon)

                                               

Questions:                                         

(1) The E.coli genome is 4.6 x 106 base pairs in length.
What is the maximum separation between two genes that is possible for them to
be co-transduced (simultaneously transduced) into a donor strain. Please explain
your answer. (5%).

           

44 kbp, this is the size of DNA packaged
in the virus. So this will be the length of the DNA. This is 0.957% of the E.
coli that can be transduced ((4.4×10^4)/(4.6×10^6))x100= 0.957%. The maximum
separation is the distance between the two genes on a homologous chromosome

                                               

(2) Suppose that you obtain the following
results in the experiment described above:-

If the culture that you used in the
experiment contained 3 x107 bacteria per millilitre, calculate the average frequency of
transduction. Explain carefully how you arrived at your answer. Do not forget
to include units. (5%)

 

(8+5+7+4+10)/5 = 6.8 = average of all
colonies

=> 1000?l/100?l= 10 =>
(3×107)/10 = 3×106 => 6.8/(3×106) = 2.27x 10-6 colonies per 100 ?l

                                   

(3) Why is it essential to be able to select
for transductants in a transduction experiment? (5%)

 

A transductant is a cell or organism that
has gone through transduction. When you select for the transductant then you
can see the bacteria that contain the gene you are looking for. Also so that you can tell the difference between Only
at low frequencies can transduction happen.

                                                           

(4) Do you think that it would be possible
to transfer a drug resistance marker from a recA mutant (defective in
generalised or homologous recombination) donor strain into a recombination
proficient recipient strain? Please explain your answer. (10 %)

           

Yes, it is possible.

(change) RecA is a protein that binds to 2
DNA molecules and aligns homologous sequences within them. It then promotes a
DNA strand-exchange reaction that creates branched DNA recombination
intermediates.

 

It is also known that the recipient strain
is not defective (only the donor strain is) therefore homologous recombination
is able to happen, therefore allowing the transfer of the marker. A recA system
is not needed as the donor strain does not take in DNA.

 

(change) Homologous recombination is
needed to transfer the drug resistant marker and two crossovers must occur. In
this case the defective recA mutant is the donor so homologous recombination
and transduction can’t happen.

 

                                               

(5) It has been observed that as the amount of
bacteriophage added to a culture increases, the number of transduction
subsequently isolated rises to a maximum level, and then declines. Provide a
hypothesis to explain these results. (10%)

 

As the number of bacteriophages (that
contain tetracycline resistance) increases therefore
increasing the number of transductions. When the optimum number of
bacteriophages has been added this will show a peak (highest number of
transductions) then the rate of transduction will decrease as cell lysis takes
place. 

 

(6) It has been noticed that the difference in frequency
of transduction between two genes can vary by almost 100-fold. Offer two hypotheses to explain this
observation. Please explain your answers carefully (15 %)

                                               

The position of the enzyme is determined
by the pac sequence, therefore the closer the gene is to the pac sequence gives
it a higher probability of being transduced.

The frequency of transduction can vary
based on the distance between the two genes, also the frequency of the gene.            

                       

                                                           

Your practical write up should include:-

                        

Write your practical up by dividing it
into the following sections: – Introduction, Results and Discussion. For this
write up ONLY, there is no need to include experimental details. Your write up
should be pitched at a level that could be understood by one of your class
mates who did not attend either the lectures or practical, but must be written
in your own words using scientific language. I expect your Introduction to
build upon the summary information given above (60% of marks for the practical
write up). Think about how the Results can best be presented. Any figures
should be properly labelled and accompanied by a legend. I would expect the
Results and Discussion sections to be relatively brief (20% each of the
remaining marks for the practical write up).

                                                           

Along with your write up, also hand in the
answers to the five questions below. The marks allocated for each question are
listed, and will count for the remaining 50% of the overall mark for the
practical.

                                               

                                   

                       

Introduction:

(lytic cycle, lysogenic cycle,
transduction, aim/objective of practical)

(fix) Transduction can be described
as “enhancing the rate of evolution in bacteria.” As the number of open reading
frames increase the relative percent of open reading frames involved in signal
transduction also increases. Prokaryotes have a short generation time and large
population sizes “One mechanism of horizontal gene transfer is transduction
which transfers DNA (genes) from one prokaryote to another.

Bacteriophage is a virus that
infects bacteria and transfers DNA from one bacterial strain to another during
transduction. Bacteriophages can replicate by two mechanisms: called the lytic
and lysogenic cycle. The lysogenic cycle allows for the replication of the bacteriophage
genome but lysis of the cell does not occur. For transduction to occur lysis
must occur so this cycle is not associated with transduction. The lytic cycle on
the other hand destroys the host cell while producing new bacteriophages
because of the lysis of the host cell. In this cycle bacteriophages connect to
specific receptor sites on an E. coli’s outer surface. The phage DNA (about 44kbp)
is then inserted into the cell which controls the production of phage proteins
and genes using the host’s enzymes and other objects within the cell. The new bacteriophages
then assembly with the genes being inserted into the capsid. The packaging
enzyme that helps in this stage does not differentiate between bacterial and
bacteriophage DNA, so defective bacteriophages (transducing particles) are formed
by bacterial genomes entering bacteriophage heads. This cycle always results in
the death of the host cell, as the cell wall breaks releasing up to 200 newly
made bacteriophages which are sent to infect healthy cells which is how the
cycle starts again and continues.

Transducing particles contain the
host DNA not the phage DNA which is inside an otherwise empty bacteriophage
head. The injected bacterial DNA must 2 homologous recombination events (cross-overs)
between the host genome and the injected DNA fragment for transduction (key in
evolution of bacteria) to actually occur. This homologous recombination is done
for exchanging DNA so the original genomic DNA is replaced by some of the
injected DNA. If a transducing particle is not homologous to the genome then it
will be part of the recipient genome therefore will be degraded by the cell.

The aim of the experiment was to
show generalized transduction, this was observed by looking at the number bacterial
colonies formed.

Pac is resembled by sequences in the
bacterial genome, these bacterial genomes are able to provide the point of
entry for the bacterial

 

 

Results:

I decided to use the class results as I do
not think that my data followed the correct pattern.

 

I then divided the total number of colonies by the total number of
plates counted to get the average number of colonies counted per plate.

Volume of lysate (?l)

Average number of
colonies (transductants) counted

0

0

10

1.06

20

1.78

30

2.56

40

2.07

50

1.06

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This data shows that as the volume of
lysate increases so does the average number of colonies counted until 30 ?l then decreases

 

 

 

 

 

 

My results:

Volume of lysate (?l)

Plate number

Total number of
colonies (transductants) counted

0

1 (control)

0

10

2

6

20

3

1

30

4

0

40

5

0

50

6

0

This table shows that as the volume of
lysate increases so does the total volume of colonies counted. For my experiments,
I used a total of 6 plates all with different concentrations.

 

 

 

 

With the
class data, many plates were used for each volume of lysate so an average was
able to be taken, while for my data only one plate was taken for each volume.
The class data is more accurate as the sample size is much larger. With my data,
an increase then decrease happens but the optimum volume occurs at a lower
volume compared to the class data. The general patter was the same but more of
my data showed that no colonies were formed, this could show that generalized transduction
had not occurred.

 

 

Discussion:

 

“why lysate
affects the number of colonies” “what could have gone better”

To
find the exact optimum volume it is important to add more values between 10 ?l and 50 ?l. An example is looking at the
average number of colonies volumes that are  4 ?l apart. A
limiting factor would be the size of the virus.

 

 

References:

·      Roca, A. and Cox, M. (1997). RecA –
an overview | ScienceDirect Topics. online Sciencedirect.com. Available at:
https://www.sciencedirect.com/topics/neuroscience/reca Accessed 21 Jan. 2018.

·      Book: Reece, Jane B., et al. Campbell Biology. ninth ed., Pearson
Education, Inc., 2014.

·      Book: Taylor, Martha
R., et al. Study
Guide for Campbell Biology, Eleventh Edition Lisa A. Urry, Michael L. Cain,
Steven A. Wasserman, Peter V. Minorsky, Jane B. Reece. Pearson, 2017.