Introduction sticky ends. (Woodhead and Malcolm, 1980) (Smith, 1996)

Introduction

 

The aim of the practical was to separate the molecules of the
negatively charged Plasmid DNA from different cultures of E. coli using Gel electrophoresis according to their size.

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The
extraction and purification of plasmid DNA from bacteria is an essential
technique in isolating the plasmid DNA from chromosomal DNA. The bacterial cell
is made up of many different components such as plasmid DNA, nucleic acids,
cytoplasmic membrane, flagella, pili and other components. Nucleic acids are
molecules that allow the transferring of genetic information. Plasmids are
small circular DNA molecules, double-stranded different from chromosomal DNA
and can replicate independently. (Neidhardt et al, 1990).

Restriction nucleases also known as restriction enzymes are
enzymes that cut DNA into fragments at specific recognition sites within the
molecules. These sites are known as restriction sites. They can be found in
bacteria, their primary function is to cleave to foreign DNA and eliminate
them. These restriction enzymes are used to manipulate the DNA and used in many
different scientific applications, such as this practical. EcoR1 (used in the
practical) is an restriction enzyme isolated from E.coli used to cut DNA in the 5′ 3′ direction to produce sticky
ends. (Woodhead and Malcolm, 1980) (Smith, 1996) (Meselson,1968).

 

Transformation of bacterial cells
is important to introduce the foreign plasmid DNA to the bacteria, allowing the
bacteria to develop the plasmid and make quantities of it. Gel electrophoresis
can then be used to separate the molecules inside the DNA into fragments
according to their size and look at how each of the different cultures.

 

Method

The
extraction and purification of plasmid DNA from bacteria is used to separate
the plasmid DNA from all other components of the cell in order to analyse it. Three
cultures were provided at the start of the practical, cultures A, B and C.

The
bacteria used was grown overnight at 37oC, the bacteria were
centrifuged to allow pellet to be pushed to the bottom of the tube, to allow
the removal of the supernatant. Solution I was then added to the pellet and mixed
together. Solution I was made up of 50mM glucose, 25mM Tris (pH8.0), 10mM EDTA
(pH8.0). It was observed here that once the Solution I was added a cloudy
precipitate was formed, this was a result of the nuclease activity of the
enzymes was inhibited and lysosome breaking down the bacterial cell wall. (Elte.prompt.hu,
2013)

Solution
II was also added to the pellet and inverted a number of times. Solution II
consisted of is 0.2M NaOH (freshly diluted from a 10M stock), 1% (w/v) SDS.
Vigorous mixing could damage the pellet and break apart the cells and ruin the
experiment, so by inverting the tube the contents are gently mixed avoiding all
possible damage. Once the pellet was re-suspended into Solution II, the
solution went from a cloudy precipitate to a clear solution. This is a result
of Alkaline lysis of the cell. The SDS disintegrated the lipid structure of the
cell membrane, denaturing both the DNA and proteins and keeping them in their
denatured form. Fatty acids and lipids are disrupted resulting in their
breakdown, forming the clear solution. (Li et al., 2007) (Ehrt and Schnappinger,
2003)

Solution
III made up of 3M potassium acetate pH 4.8 was then added to the pellet. Due to
the potassium salt being insoluble, the solution went from clear to form a cloudy
precipitate, the precipitate formed was made up of the dissolved proteins and
the plasmid DNA, making the plasmids to be more visible.

The
centrifugation of the Eppendorf tube allowed all the chromosomal liquid to be
removed from within the pellet. “95% ethanol” was added to the pellet and immediately
discarded, “70% ethanol” was also added and discarded immediately. The addition
of the ethanol resulted in the precipitation of the plasmid DNA, the
precipitate was washed in “70 % ethanol” to remove all the excess salt content
inside of the pellet. Specifically, “70 % ethanol” is used to allow water
molecules to be retained in the pellet, in order to make the solution soluble. (Oswald,
n.d.)

Then
pellet was then dissolved in a solution of TE. TE is made up of 10mM Tris
(pH7.6), 1mM EDTA (pH8.0).  A mixture of
Culture B and RNA’ase was added to a new tube to form Tube B+.

Restriction
nucleases were used to digest Culture B and C to form the new solutions Tube BR
and Tube CR. The components of the Tubes BR and CR are listed below and were
added in the order written.

Tube
BR:

•                   
8
µl of Water

•                   
2
µl of 10 x EcoR1 Buffer

•                   
5
µl of DNA B

•                   
5
µl of EcoR1 enzyme

 

Tube
CR:         

•                   
8
µl of Water

•                   
2
µl of 10 x EcoR1 Buffer

•                   
5
µl of DNA C

•                   
5
µl of EcoR1 enzyme

Water
and buffer were first added to the tube to avoid the denaturing of the enzyme.
Although buffer is supposed to help stabilise the pH of the enzyme, if the concentration
is too high the enzyme may be risk denaturing as a result of the ionic
strength. Addition of water to the buffer helps to make the environment more
acceptable for the enzyme. (Bisswanger,2014)

The bacterial cells were left overnight
in L-broth and shaken at 37oC. Competent cells are cells that can
accept plasmids from the environment. They can occur naturally or artificially
through a chemical treatment called Calcium Chloride Transformation, these
cells are used in order to make them momentarily permeable to DNA, they have
also been recognised to have a higher transformation efficiency. (Aich et al,
2012)

 

The cells were kept in an ice
bucket in order to increase the maximum transformation efficiency, if the cells
were to reach a temperature above 0oC, the transformation efficiency
could decrease and the experiment will be comprised.

 

After centrifugation of the cells,
CaCl2 was added to the cells and the supernatant discarded, once the
cells were removed from the ice bucket. Culture B was diluted with water and
labelled “Diluted B” in a new Eppendorf tube. Three more Eppendorf tubes were prepared;
their components are listed below:

 

•          Tube
1: 100 µl of 0.1M Tris pH 7.6

•          Tube
2: 2 µl of “diluted B” and 98 µl of 0.1M Tris pH 7.6

•          Tube
3: 2 µl of Tube BR (EcoR1 digested B) with 98 µl of 0.1M Tris pH 7.6.

 

“Diluted B” was added to each of
the three tubes and left in the ice bucket before being transferred to a water
bath. Once the tubes were removed, they were placed in the ice bucket before being
suspended in L-broth and then spread on LB-amp plate. Lysogeny broth is a medium
rich in nutrition primarily used in encouraging the growth of bacteria. The
cells were grown overnight at 37oC.

 

Gel Electrophoresis is used for
separating proteins, DNA and RNA according to their size and charges. Smaller
molecules travel through the gel faster, while larger molecules travel at a
slower rate. Fluorescent / radioactive dyes are used to allow the DNA to be
seen after it has been separated (these form the bands on the gel). Syber Safe
is a cyanine dye used a DNA stain for identifying DNA in agarose gels by
binding to DNA, it acted as a loading buffer.

The comparison of the bands formed
on the gel, allows you to work out the approximate length of the DNA fragments.

 

The agarose gel was prepared using
molten gel (1% w/v in 1x TBE buffer). The gel was poured into and former with “combs”
and allowed to set. 5 µl of solutions A, B, C, B+, B/EcoR1 and C/EcoR1 were mixed
with 10 µl of TE Buffer and 5 µl of Loading Buffer to form solutions.

 

Once the samples were mixed and centrifuged
they were each loaded into the agarose gel with Marker X (blue dye). An
electric current was also applied to the gel. Once the blue dye had run the gel
was turned off and looked at using an trans illuminator. The electric current
applied across the gel allowed one side to be positively charged and other end
to be negatively charged. DNA is negatively charged, so it will migrate to the
positively charged area of the gel and the will separate according to size.
(Alberts,2002)

The TBE buffer was made up of 90mM Tris-borate and 2mM EDTA. The Loading
buffer was made up of 0.25%n bromophenol blue and 15% Ficoll 400.

 

 

Results

 

Tube 1 was made up of 100 µl of 0.1M Tris pH 7.6 and 1 m L of
L-broth. 100 µl of the solution was then taken and added to an agar plate –
this formed Treatment 1. On the agar plate, 0 colonies were observed, since
there was DNA on the plate. The L-Broth acted as the active agent- nutritionally
rich for growth of bacteria.  

 

Treatment 2 was made of 100 µl of 2 µl of “diluted B” and 98
µl of 0.1M Tris pH 7.6 with 100 µl of L-Broth placed on an agar plate. On the
plate, 60 colonies were observed to have formed instead of a supposed 300
colonies. The plasmid DNA from the “Diluted B” was supposed to have taken up
and digested the nutrients from the L-Broth and grown significantly. The colonies
observed consisted of large colonies surrounded by smaller colonies. These halo
surrounding colonies suggest that the colonies were incubated for too long.

 

Treatment 3 was made up of 2 µl of Tube BR (EcoR1 digested B) with
98 µl of 0.1M Tris pH 7.6 with an additional 100 µl of L-Broth. On the plate, 0
colonies formed in comparison to a supposed 3 colonies that should have formed.In gel electrophoresis, the plasmid DNA can exist in three
different forms – these include: supercoiled, open-circular (oc) and linear.
The migration of the plasmid DNA in Figure 1 was from top to bottom. Smaller
molecules are known to pass through the gel much quicker than larger
molecules.  Many tracks within the image
contain smears, this can be a result of the sample not being prepared properly
as wells as overloading of the well.  In Figure 1, the ladder above Track M goes up from 0.1 kilobase
pair (kbp) to 10.0 kbp. The bands go in order of the lowest to highest. Track M
shows of the ladder of linear pieces of DNA. The bands get brighter as the kbp
increases, it is clear that the Marker worked well as the DNA bands gets closer
to each other.  Track A had no DNA, the culture only had RNA inside. The smear in
Track A, goes in order of rRNA, mRNA and tRNA with tRNA being at the bottom.
This suggests that tRNA was the smallest molecule, as it was at the bottom of
the smear, indicating it passed through the gel first. The intensity of the
smear is about the same from top to bottom, suggesting that the fragments are
present in equal amounts.  In Track B, there is a smear which consists of RNA. The band above
the smear is a linear plasmid at around 2 kbp. It is clear in Track B, that the
fragments are present in unequal amounts as the intensity of the fragments
differ, the intensity increases as the kpb becomes lower.  Track C has a variation of supercoiled, linear and open-circular
plasmids. The band above the smear (6.0 – 8.0 kbp) has a very high intensity in
comparison to the rest bands in Track C. Again, it is clear that all the
fragments were generated by digestion of more than one different molecule as
they are represented in unequal amounts. At the very top of Track C, there is a
visible band, which appears to have gotten stuck in the well, this could be due
to the fact that the molecule was too big or it didn’t have enough time to run
through the gel.  In Track B+, there are only two visible fragments, the fragment
around 2.0 -3.0 kbp is supercoiled with a very high intensity. The RNA runs as
multiple bands, however once it was treated with ribonuclease it disappears,
the ribonuclease contaminated everything by removing RNA, which is why there
are no other visible fragments below the supercoiled fragment.  Track B/R1 only has one visible fragment – a linear piece of DNA
with a high intensity at 3.0 kbp and one EcoR1 site. Only one band is seen
above the track this is observation is correct, as the sample has been
restricted with EcoR1. This is due to the fact that each plasmid has a unique
restriction site where the restriction enzyme should cut it to produce
restriction fragments.   

There were three visible bands in Track C/R1. The intensity of the
fragments decreased significantly after the two fragments between 4.0 and 6.0
kbp. At 8.0 kbp the fragment with the less intensity can be identified as a
Digestive Enzyme, so it was single digestion with multiple partial digestions
which aren’t visible. This shows sign of contamination at some point during the
gel electrophoresis. Track C/R1 was also found to contain two EcoR1 restriction
sites, this is important because EcoR1 should have recognised the two sequences
in the plasmid DNA and cut it to produce the fragments. However, there are
three bands above the Track instead of two. This suggests that either the
plasmid was partially cut or the restriction enzyme was damaged.