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Transduction
Námsbraut: Raunvísindadeild; Líffræðiskor
Námskeið: 09.51.35 Erfðafræði
Nafn kennara: Ólafur S. Andrésson, Zophonías O. Jónsson, Sigríður H. Þorbjarnardóttir og
Bryndís K. Gísladóttir.
Vikudagur, hópur: Fimmtudagur, síðari kennslustund, Hópur 2 og 4.
Tilraun framkvæmd: 18., 25. september, og 2., 9. október, 2003
Skýrsluskil til kennara: 30. október, 2003
Skýrsluskil til stúdents:
Einkunn:
Nöfn stúdenta: Bjarki Steinn Traustason, Egill Guðmundsson, J. Gabriel-Rios Kristjánsson,
Marcella Manerba og Nicoletta Palmegiani.
________________________________
Bjarki Steinn Traustason
________________________________
J. Gabriel-Rios Kristjánsson
________________________________
Egill Guðmundsson
________________________________
Marcella Manerba
________________________________
Nicoletta Palmegiani
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Raunvísindadeild; Líffræðiskor
6
Erfðafræði
Transduction
Introduction:
In this process, a bacterial DNA fragment is transferred from one bacterial cell to
another by a phage particle containing the bacterial DNA. Such a particle is called a
transducing phage. There are two types of transduction, general and specific. In the
former type, any part of the bacterial genome can enter the virus particle, but has to be
of the same length as viral DNA. The latter type is more specific because only certain
can be transferred into the viral particle.
During this experiment we used a generalized transducing phage, P1 virus,
that produces some particles that contain DNA obtained from the host bacterium, E.
coli (CGSC1255), rather than phage DNA; this DNA fragment can be derived from
any part of the bacterial chromosome.
Following infections, the virus’s DNA, can either inhabit the E. coli cells as a
plasmid and replicate, according to the bacterial chromosome, and stay inactive for
generation after generation (lysogenic cycle), or it can proliferate and causes bacterial
lysis (lysis cycle).
During the infection by P1, the phage makes a endonuclease that cuts the bacterial
DNA into fragments (~100 kb) and this are occasionally packaged into phages
particles instead ‘place’ of P1 DNA.
The position of the nuclease cuts in the host chromosome are random, so a
transducing particle, may contain a fragment derived from any region of the host
DNA. When a transducing particle adsorbs to a bacterium, the bacterial DNA
contained in the phage head is injected into the cell and becomes available for
recombinations, as a part or as a whole.
The probability that a particular gene will be carried in a phage particle can be
calculated from the frequency of transducing particles (0.003) and the fraction of the
bacterial chromosome contained in such a particle (0.024). This probability is about 6
∙ 10-5.
Generalized transduction allow us to derive linkage information about
bacterial gene, when markers are close enough, the phage can pick up and transduce
them in a single piece of DNA (cotransduction). Linkage values are initially expressed
as a cotransduction frequences, → the greater this frequency is, the closer two genetic
markers are.
In 1966, T.T. Wu discovered a mathematical expression, in which frequency of
transduction, x, and distance of mapping are related to each others:
d = L ( 1 – 3√x )
d= distance between two gene [min].
L= length of chromosome carried by phage during transduction. For P1 this length is
about 2% of the bacterial chromosome, similar therefore, to 2.1 min. In fact any
markers separated by more than 2.1 min will be not cotransduced by P1.
There are three main differences between transduction and conjugation. (1)
The nature of these processes is different, because, the first occurs between two
bacterial cells, while the second occurs between bacterial cell and a virus. (2)
Transduction is rare and a random process. Conjugation is specific, and always occurs
when there are is an F-factor involved. (3) Conjugation can transfer, from a donor to a
recipient cell, almost all of his genome, while only 2 - 2.5% of the genome can be
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transferred during transduction. That is the reason why conjugation is used to
determine the relative order and distance between genes far from each other in the
chromosome. On the other hand, transduction is used to map gene really close to one
another.
Aims/hypothesis:
The aim is to map Tn10-transposable genetic element (transposone) considering
argH1 and metB1.
Design and Methods:
Reference to work sheets in manual booklet, for present exrecise (p.25-28).
Exception in the procedure part, p.28, where the sediment was dissolved in
500 µL of buffer, but not 1 mL.
Results:
TABLE 01, NUMBER OF COLONIES PER PETRI PLATE WITH DIFFERENT TYPES OF MEDIUM:
Medium, types:
Group:
H1
H2
H3
H4*
H5
Met–
Arg–
L-Tet
156
243
183
36
172
182
223
224
28
151
63
143
129
20
95
186
195
108
* These petri plates were incubated with greater dilution, a dilution not known, as a mistake. Given that
clarification, these numbers were not taken into count for mean value, so that mean difference between numbers of
colonies in different medium deported more clearly.
mean
TABLE 02, CONTROL PLATES; NUMBER OF COLONIES PER PETRI PLATE WITH DIFFERENT TYPES OF MEDIUM:
Medium, types:
Strain:
1255
P118500
Met–
Arg–
L-Tet
0
0
0
0
0
0
mean
0
0
0
TABLE 03, REPLICA PLATING WITH Met+-BACTERIA; NUMBERS OF COLONIES PER PETRI PLATES WITH
DIFFERENT TYEPS OF MEDIUM:
Group:
H1
H2
H3
H4
H5
Σ
Met+ TetR Arg+
Met+ TetR Arg–
Met+ TetS Arg+
Met+ TetS Arg–
4
0
37
9
2
0
25
23
1
0
33
16
2
0
18
30
2
0
32
16
11
0
145
94
50
50
50
50
50
250
Σ
TABLE 03, REPLICA PLATING WITH Arg+-BACTERIA; NUMBERS OF COLONIES PER PETRI PLATES WITH
DIFFERENT TYEPS OF MEDIUM:
Group:
H1
H2
H3
H4
H5
Σ
Arg+ TetR Met+
Arg+ TetR Met–
Arg+ TetS Met+
Arg+ TetS Met–
1
6
17
26
2
2
26
20
6
2
9
33
2
5
18
25
1
7
18
24
12
22
110
128
50
50
50
50
50
250
Σ
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TABLE 04, REPLICA PLATING WITH TetR-BACTERIA; NUMBERS OF COLONIES PER PETRI PLATES WITH
DIFFERENT TYEPS OF MEDIUM:
Group:
H1
H2
H3
H4
H5
Σ
TetR Arg+ Met+
TetR Arg+ Met–
TetR Arg– Met+
TetR Arg– Met–
2
16
0
32
3
13
3
31
5
18
0
27
4
13
1
32
11
11
0
28
25
71
4
150
50
50
50
50
50
250
Σ
TABLE 05, COTRANSDUCTION FREQUENCE ACCORDING TO REPLICA PLATING, IN PERCENTAGE [%]:
+
Met
Arg+
TetR
TetR
Arg+
Met+
4.4
13.6
100.0
62.4
100.0
38.4
100.0
48.8
11.6
FORMULA 01, CALCULATIONS FOR COTRANSDUCTION FREQUENCY:
General formula:
Considering gene a-medium, with gene a, b, involved.
(y1/Y) = x
x : the ratio, cotransduction frequence
y1 : total number of colonies that are a+ b+.
Y : total number af all colonies, without considering special medium.
Concidering Met–-medium, with the gene Met, and, Tet, involved.
((11 colonies + 0 colonies)/250 colonies) = 0.044
Eg,:
TABLE 06, DISTANCE BETWEEN TWO GENES [min]:
TetR
Arg+
Met+
+
Met
Arg+
TetR
1.36
0.31
0.00
1.08
0.00
0.57
0.00
0.45
1.02
As the ratio gets larger for two genes, the distance between them becomes more less.
The order is TetR, Arg+, Met+.
FORMULA 02, CALCULATIONS FOR DISTANCE:
General formula:
d = L ( 1 – 3√x )
Eg,:
x : the ratio, cotransduction frequence
L : length of chromosome carried by phage during transduction.
Here, for P1, it is 2.1 min.
d : distance between the two genes [min].
2.1 min (1 – (0.044)1/3) = 1.36 min
FIGURE 01, THE GENETIC MAP:
R
Tet
———————————
d1, between TetR and Arg+
(1.08 min + 0.45 min)/2
= 0.76 min
= 46 sec
Arg+
———————————
d2, between Arg+ and Met+
(0.31 min + 0.57)/2
= 0.44 min
= 26 sec
d3, between TetR and Met+
(1.02 min + 1.36 min)/2
= 1.19 min
= 1 min 11 sec
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Met+
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Conclusion/Discussion:
According to cotransduction frequency (TABLE 05), the frecuency from Arg+ to Met+
is comparable to frequency from Met+ to Arg+, so cotransduction is successful in both
directions. On the other hand, the frequency from TetR to Arg+ or Met+ are not that
comparable to the frequency from Arg+ or Met+ to TetR. This difference is accounted
to TetR-gene, which is a transposable genetic element, and has no complementary
location on chromosome 1255.
■
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