MICR3003
Practical Report 1
Location of Mitotic Proteins in
Escherichia
Coli
Cell
DivisionIdentification using GFP
Katherine Jordan
Abstract
The location of several vital mitotic proteins, FtsZ, FtsK and MinD, were examined in the
Escherichia coli strain JM109 using Green Fluorescent Protein (GFP). Mitosis is the process
by which bacteria replicate themselves and produce ‘daughter cells’, which involves many
stages. FtsK is vital in the separation of material within the cells, MinD organizes the cells
before separation, and FtsZ and MinD are involved in separating the two daughter cells.
The three genes that encode the proteins were inserted into small strands of DNA called
plasmids and linked with GFP, before the plasmids were inserted into the E.coli cells using
increased Calcium levels and heat shock.
When the cells expressing these proteins were examined under blue light using an Olympus
BX51 microscope, FtsZ was found in bands around the centre of the cell and MinD was
found at the poles. Both of these results were expected. The location of FtsK could not be
determined, as the gene was not fully inserted into the plasmid due to its length. The cells in
which the proteins were overexpressed were also elongated. This is believed to be because
the proteins trigger more mitotic events than the cell can keep up with. This study adds more
knowledge to the process of mitosis.
Introduction
Bacterial replication is achieved by a process known as mitosis, a duplication of cellular
material that is then divided into two distinct but identical ‘daughter cells’. Mitosis is a well
understood process, and the proteins involved and their functions are well documented.
One way to examine proteins is by using Green Fluorescent Protein, or GFP, which is often
used to identify the locations of protein within the cell. GFP glows green when exposed to
blue light, and by linking it to another protein, allowing expression of the joined proteins and
exposing the containing cell to blue light, the location of the target protein can be seen
through a specialized microscope.
There are many proteins involved in cell division, including FtsZ, FtsK and MinD. During
mitosis, chromosomes are duplicated and then segregated. FtsK is linked to the cell wall and
is highly involved in the segregation and timing of this process (Weiss, 2004). MinD forms
extensive filamentous structures in the cell to allow organization, and also recruits MinC and
MinE, a complex which controls where the Z ring, a ring of proteins that constricts during
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cell division and separates the daughter cells, forms by localizing in the poles of the cell and
preventing its formation in this area (Ebersbach and Gerdes, 2005). FtsZ is responsible for the
formation of the Z ring (Jennings et al., 2011).
The aim of this experiment was to use GFP to determine the location of these proteins inside
E. coli cells. It was believed that FtsZ would be centralised around the centre of the cell to
initiate division, FtsK would also be located in the middle of the cell to separate
chromosomes, and MinD would be found at the poles of the dividing cell. Each gene was
inserted into a GFP-containing plasmid (pGFPuv), a small section of functional DNA that can
be inserted into bacteria and expressed, in a way that will allow the two genes to be coexpressed. The plasmid also contained an Ampicillin resistant gene to allow the cells
containing the plasmid to be selected for in an ampicillin rich medium. The plasmids were
introduced into Escherichia coli JM109 colonies and examined using fluorescence
microscopy.
Materials and Methods
To insert the target genes into the pGFPuv plasmid (containing an ampicillin resistance gene),
large samples of both were obtained by a Polymerase Chain Reaction (PCR), the process of
which allowed XbaI and HindIII recognition splice sites to be added to both. Gel
electrophoresis and a UV transilluminator were used to find exact concentrations of the
plasmids, and the enzymes XbaI and HindIII were used to cut the samples at the recognition
sites. A NanoDrop Spectrophotometer was used to find the concentration of the proteins,
purified using the Wizard® SV Gel and PCR Clean-Up System before the proteins were
inserted into the plasmid and secured using T4 DNA ligase.
The E.coli cells were made more permeable using high levels of calcium, allowing the
insertion of plasmids with the heat shock technique. The cells were placed onto 6 nutrientrich and ampicillin-containing agar plates and incubated overnight: 1 with plasmids with no
gene, 1 with cut plasmids with no gene, 1 with pure H2O and 3 with cells containing 1 of
each of the 3 genes. The Quantum Prep® Plasmid Miniprep Kit allowed isolation of plasmids
from the cells, and the presence of plasmids was tested for using restriction digestion and gel
electrophoresis.
Expression of the target proteins was induced using Isopropyl-β-D-thiogalactoside (IPTG)
and cells were incubated at 37ºC on a shaker for 60 mins. A positive control containing only
the GFP protein was included, and a negative control was not induced with IPTG. Samples of
each of the induced cells were then placed on a slide and examined for GFP expression under
blue light using an Olympus BX51 microscope.
Results
The insertion of different plasmids into the cells resulted in varying numbers of viable
colonies being produced on the agar plates (Figure 1).
Plate Number
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Plate Name
Colonies (approx.)
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1
2
3
4
5
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Practical Report 1
Positive control (undigested pGFPuv)
Control (digested pGFPuv)
Negative Control (No DNA)
pGFPuv-FtsZ ligation mix
pGFPuv-FtsK ligation mix
pGFPuv-MinD ligation mix
1040
57
0
220
242
340
Figure 1. Approximate number of colonies obtained from allowing cells containing variously altered pGFPuv plasmids to
grow overnight in agar plates containing ampicillin.
The plasmids were shown to be present in all of the cells by restriction digestion and gel
electrophoresis (Not shown). It was found that the FtsZ protein appears in bands around
multiple areas of the E.coli cells, and that the modified cells were also vastly elongated (See
Figure 2). MinD proteins were found at the poles of the cell, and were likewise elongated
(See Figure 3). FtsK plasmid cells did not show any fluorescence and were not elongated, and
the positive control cells containing only the plasmid showed fluorescence but not elongation.
Figure 2. Artists Representation of FtsZ location
in cells
Figure 3. Artists Representation of MinD
location in cells
Discussion
The proteins were expected to be expressed in certain areas within the cell. The role of FtsZ
in the Z ring formation should have seen this protein in the center of the cell (Jennings et al.,
2011), and the role of MinD in the regulation of its placement should have seen it at the poles
of the cell (Ebersbach and Gerdes, 2005). This proved to be the case. FtsK should also have
appeared in the centre of the cells due to its part in chromosome segregation (Weiss, 2004,
but due to the FtsK protein not being expressed in the plasmid, its location could not be
determined.
The cells with the insert were shown to be unnaturally elongated. This is believed to be a
result of the increased amount of inserted protein preparing the cell for more divisions than
the normal levels of other proteins in the cell can keep up with, suggesting that FtsZ and
MinD have a role in initiating cell division.
The ftsK gene is very long and therefore difficult to insert into plasmids. Tests showed that
only the first half of the gene was actually present in the cell, and as such, no FtsK-GFP
protein was produced.
The controls plated after the plasmids were inserted were there for various reasons. Plate 1,
the positive control, was to determine that the cells used in the experiment were actually
viable, and that the plasmid was being inserted correctly. There was a large number of cells in
this colony, showing that the cells were viable. If the number of colonies in plates 1 and 2
were approximately the same, it would show the plasmid had self-ligated and inserted itself
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into the cell. This would mean that some of the plasmids may not contain the genes they are
supposed to, and may not produce the desired proteins. Plate 2 should have no colonies
growing, but not all of the plasmids would have been cut properly, and because the protein
plates- 4, 5 and 6- contained more colonies than plate 2, it can be assumed the cells are not
self-ligating. Plate 3, containing no plasmid DNA and therefore no antibiotic resistance gene,
was run in order to determine whether the cells were naturally immune to ampicillin. Had
Plate 3 grown colonies, the ampicillin based selection of cells containing plasmids had not
worked, and it could not be assumed that all of the cells in any of the plates contained the
plasmid.
The positive control in the final stage of experimentation before the cells were examined
under the microscope was run to ensure the GFP gene used in the experiment was
functioning. Had these positive control cells not lit up, it would show the GFP gene was
faulty. Had no fluorescence been shown in the other cells, but had been shown in this cell, it
would be concluded that the insertion of the proteins had not worked.
The negative control was to ensure that the plasmids had not been activated before they were
induced, and to ensure that an equal amount of time was given for all the cells to produce
their proteins.
This experiment has determined the locations of several cell division-related proteins in cells
undergoing mitosis.
Bibliography
Ebersbach, G., Gerdes, K. (2005) Plasmid Segregation Mechanisms. Annual Review of
Genetics 39: 453-479
Jennings, P.C., Cox, G.C., Monahan, L.G., Harry, E.J. (2011) Super-resolution imaging of
the bacterial cytokinetic protein FtsZ. Micron 42: 336-341
Weiss, D.S. (2004) Bacterial cell division and the septal ring. Molecular Microbiology 54:
588-597
Paper title: SCIE3001 assignment rewrite
Paper ID: 345211572
Author: Katherine Jordan
Katherine Jordan
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