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Conjugation in E. coli
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. október, 2003
Skýrsluskil til kennara: 16. 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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Conjugation in E. coli
Introduction:
E. coli’s chromosome is one continuous DNA-molecule, about 1.3 mm on length.
In the cytoplasm of some E. coli-strains, is a so-called F-factor which is a small
circular DNA molecule which goes under replication independent to the chromosome’s replication. The Bacteria which have the F-factor are called F+, but the ones
without it are called F–. The F-factor encourages to conjugation between F+- and F–bacteria, when they are cultured together. At conjugation, the F-factor transfers into
the F–-bacterium and it will become F+. The (first) F+-bacterium retains at least one
copy of the F-factor and remains F+.
Once in a while (in about, every (other) 105 cell) recombination occurs
between the F-factor and the bacterium’s chromosome. During conjugation, a part of
the chromosome can transfer to the F–-cell along with the F-factor. In Hfr-strains, the
F-factor has integrated into the bacterium’s chromosome and persisted there for
generation after generation. In these strains, each and every cell can transfer it’s
genetic material if it reaches conjugation with F–-cell.
Hfr-cell traslocates it’s genes in certain order at the same time the
chromosome is replicated. Replication and translocation starts in certain ‘set-point’ in
the integrated F-factor and can continue until the whole chromosome has been
transferred, and the F-factor amongst it. The translocation of the whole chromosome
takes about 100 min at 37 °C, but the connection (in conjugation) between Hfr- and F–
-cell frequently rupture/uncouple before the translocation for the whole chromosome
has concluded. After the genes from F+- or Hfr-cell have been transferred into F–-cell,
they can recombinate into the F–-chromosome. The gene which have been
incorporated, inherit to the next generation but the others are destroyed.
In this exercise, we monitor gene translocation from E. coli-Hfr cells
(CGSC6026) to E. coli-F– cells (CGSC1157). The F–-strain is auxotroph, concerning
the amino acids, threonine, leucine, proline, and histidine, but it is, however, resistant
to streptomycin, because it has the rpsL mutation in ‘ribosome protein’-gene. The
Hfr-strain is streptomycin-sensitive, which is a dominant feature. By incubating on
various medium, you can see a gradient figure in the number of colonies which grow,
reckon with the genes’ loci in the chromosome, which was transferred to the recipient.
Abbreviations: E. coli, Escherichia coli; F-factor, fertility factor; Gal, galactose; His, histidine; Hfr, high frequency
of recombination; Leu, leucine; Pro, proline; Thr, threonine.
Aims/hypothesis:
The aim, is to determine bacterial conjugation, the order of gene translocation from
Hfr-E. coli strains to F–-strains, and to localise genes on E. coli’s chromosome.
Design and Methods:
Reference to work sheets in manual booklet, for present exrecise (p.18-20). Exception
from the 6th part, p.20, where petri dishes were incubated with 0.1 mL of every
dilution. Used dilutions were: for pro–-dishes, incubated separately with 10–3 and 10–4
dilutions; instead of 10–2 and 10–3 dilutions.
Using more dilute solutions makes the counting of colonies easier, which leads
up to more intuitive viable count.
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Results:
TABLE 01, E. COLI’s VIABLE COUNT IN DIFFERENT MEDIUM:
pro+
type:
colonies
dilution:
Group:
H1
H2
H3
H4
H5
(thr/leu)+
his+
colonies
colonies
10–3
/dish
10–4
viable c.
10–2
/dish
10–3
viable c.
10–1
/dish
10–2
viable c.
173
192
220
186
296
10
17
29
34
31
1.7106
1.9106
2.2106
1.9106
3.0106
828
-
273
192
205
280
362
2.8106
1.9106
2.1106
2.8106
3.6106
11
281
126
9
284
1
5
0
0
10
1.1103
5.0103
1.3103
9.0102
1.0104
2.1106
2.6106
3.7103
Viable count, general formula: (colonies ∙ (volume of solution / volume used for incubation)) / dilution.
Eg,: viable count = (192 colonies ∙ (1.0 µL solution / 0.1 µL used for incubation)) / 10–3 = 1.9 ∙ 106.
<viable count>:
TABLE 02, CONTROLES, NUMBER OF COLONIES PER PETRI DISH:
type:
strain:
6026
1157
pro+
(thr/leu)+
his+
0
0
0
1
0
0
TABLE 03, PARALELLISM OF COLONIES IN DIFFERENT MEDIUM, AFTER REPLICA PLATING:
Group:
pro+ gal+ his+
pro+ gal+ his–
pro+ gal– his–
pro– gal+ his+
pro– gal– his+
pro– gal+ his–
pro– gal– his–
Σ
H1
H2
H3
H4
H5
Σ
1
3
24
1
0
2
19
50
2
8
17
0
0
0
23
50
1
4
19
1
0
0
25
50
1
7
21
1
1
0
19
50
2
7
22
0
0
2
17
50
7
29
103
3
1
4
103
250
TABLE 04, NUMBER OF COLONIES PER PETRI DISH, WHICH GROW AFTER REPLICA PLATING:
type:
Group:
H1
H2
H3
H4
H5
Σ
pro+
gal+
his+
28
27
24
29
31
139
7
10
6
9
11
43
2
2
2
3
2
11
TABLE 05, THE RATIO FOR THE TYPES OF COLONIES:
Total, from H2
type:
pro+
:
gal+
:
his+
:
( 27/250) ∙ 100% = 54.0%
( 10/250) ∙ 100% = 20.0%
( 2/250) ∙ 100% = 4.0%
Total, from H4
type:
pro+
:
gal+
:
his+
:
( 29/250) ∙ 100% = 58.0%
(
(
9/250) ∙ 100% = 18.0%
3/250) ∙ 100% = 6.0%
... in comparison
Total, from all groups
type:
pro+
:
(139/250) ∙ 100% = 55.6%
gal+
:
( 43/250) ∙ 100% = 17.2%
his+
:
( 11/250) ∙ 100% = 4.4%
general formula: (the total number of specific type of colonies / the total number of all types of colonies) ∙ 100%
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FIGURE 01, DETERMINATION OF THE LOCATION OF THE proA-GENE AND THE galK-GENE, COMPARED WITH
THE EQUATION OF THE COLONY-FUNCTION:
Determ. of proA:
From Table 04, y = 139 (ie,
the total number of colonies,
type pro+).
The x-value is the unknown,
and locates the minutes.
Determination of proA
number of colonies
1000
y = 209.93e-0,0703x
100
10
1
0
10
20
30
40
50
t [min]
Determ. of galK:
From Table 04, y = 43 (ie, the
total number of colonies, type
pro+).
The x-value is the unknown,
and locates the minutes.
Determination of galK
number of colonies
1000
y = 240.25e-0,0704x
100
10
1
0
10
20
30
40
t [min]
y = 209.93 · e–0.0703x
; y/209.93 = e–0.0703x
; ln(y/209.93) = –0.0703x
; x = ln(y/209.93) / –0.0703
; x = ln(139/209.93) / –0.0703
x = 5.8649
x~6
50
y = 240.25 · e–0.0704x
; y/240.25 = e–0.0704x
; ln(y/240.25) = –0.0704x
; x = ln(y/240.25) / –0.0704
; x = ln(43/240.25) / –0.0704
x = 24.439
x ~ 24
Conclusion/Discussion:
The colonies which grow on the agars, are only the F–-strains, through conjugation
have gotten their genes, which allow the biosynthesis of the amino acids: threonine,
leucine, proline, histidine. Hfr-strains don’t grow in the agar because it contains
streptomycin, which the bacteria are sensitive to.
The control dishes show that the strains are correctly defined, ie, the Hfrbacteria are streptomycin sensitive and the F–-strain is auxotroph that needs the amino
acids above-mentioned to grow. Because of the Hfr-strain is streptomycin sensitive
we shouldn’t expect any colonies to grow on the dishes. From Table 02 ■, evidently
one colony grew in one of the control dishes, which is plausibly contamination,
judging from it appearance.
Estimating from our numbers of colonies that can grow on different agars,
and our given temporal order of inheritance, we can guestimate were the F-factor was
incorporated into the recipient’s chromosome. Based on the higest average data of
viable count, Table 01 ■, we can assume that the F-factor was integrated upstream to
the thr/leu-genes. In fact those genes adjacent to O (origin to translation) are
transferred first, leading the mobilized chromosome into the F–-bacteria. The F-factor
therefore becomes the end of the broken chromosome, and is the last part to be
transferred. This proposal explains why the recipient cell, when conjugated with Hfr,
remains F–.
The second highest mean of viable count came from the colonies which grew
on the pro–-agar, and lowest mean of viable count was on the his–-agar. Ie, the
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assumed order is: thr/leu → pro → his. The Ratio given in Table 05 ■, also confirms
the order, in addition to, applying galK into the order. The translocation of the
genome starts at 98th min in Hfr-strains and it is likely that the F-factor has integrated
near that location.
Answers to Questions:
1.
In what part of the chromosome is the F-factor integrated, in Hfr-strain CGSC6026?
The F-factor has integrated near that location at 98th min.
2.
Check the results from the petri dishes, which were incubated after conjugation. What is the
appearent order of gene translocation?
The assumed order is thr/leu → pro → his.
3.
Observe data resulting from using replica plating-method. What is the most likely order of the
genes? Is the order the same as data from incubation after conjugation dictate?
Our results confirm the given order of the genes.
4.
Why aren’t all pro+-blots also (thr/leu)+?
Sometimes recombination occurs in the chromosome between thr-gene and
leu-gene so they do not come through, which means that some colonies can be
pro+ and (thr/leu)– at the same time.
5.
What is the function of the controls?
The control dishes show that the strains are correctly defined.
6.
If the location of leuB, his, and galK is given, on what minute does proA localise, according to
frequency-data from replica plating?
According to graphic calculations, proA is on 5.86th min, ~ 6th min. Figure 01 ■.
7.
How did the mapping of galK come along?
According to graphic calculations, galK is on 24.4th min, which is far from the
right value, 17th min, Figure 01 ■. When we calculated the position of the hisgen we got a value which was also far from the right value. This probably due
to the long distance from the origin of the translation. The error is also evident
by comparison of the line-equations, focusing on different slope’s values.
8.
How do your results conform with what is known, by contrast to, eg, information on p.17 and
18 in manual booklet?
The regularisation is good.
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