Universidade de Sao Paulo

Thiago Tarraf Varella, n 8993646

Biodiversity at a lacustrine environment

Genetic analysis with samples from a lake at Minas Gerais, Brazil

CCM0215 Report
Molecular Biology Laboratory
Molecular Sciences Undergraduate Program
Universidade de Sao Paulo (USP)

Under the guidance of:

Profa. Dra. Marie-Anne Van Sluys.
Instructors:
Msc. Paula Turrini
Dra. Tatiana C. S. Corra

Sao Paulo
June 2015

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1
1.1

Introduction
Biodiversity

The Earth is estimated to be more then 4 billion years old[2] , and over the years it is
known that the organisms turned more and more complex. Many biological processes
started to happen and, concomitantly, the creatures have become increasingly distinct.
The most recent classification of all existant organisms is around three Domains of Life,
proposed at first by Carl Woese in 1990: Archea, Bacteria and Eukarya[3] .
Archea is a domain composed by prokaryotic cells, usually containing membranes
with glycerol attached at branched hidrocarbon chains by ether linkages. Bacteria, just
like Archea, is composed by prokaryotic cells, but with a different membrane: there
are unbranched fatty acids chains attached to glycerol by ester linkages. Eukaryans are
composed by cells containing nucleus and other organelles enclosed within membranes.

1.2

Genetic Code

Deoxyribonucleic acid, or DNA, is a polymer forming a double helix with a backbone

of sugar and phosphate. This backbone is attached to a sequence of 4 different types of
bases: adenine (A), guanine (G), cytosine (C) and thymine (T). The sequence of the
bases forms what is called the genetic code, a hereditary material possible due to the
fact that the DNA is able replicate. This hereditary material gives information so that
the code can be translated into proteins and promote a significant part of the processes
of almost all living beings.
The ribosomal DNA is a fragment that code the ribosomal RNA, a component of
the ribosome. The conservation of the ribosomal DNA is different for each domain
of life (Isenbarger et al., 2008)[4] . With this information, it is possible to classify the
different specimens at different empires only be manipulating its genetic code.

1.3

Computational Molecular Biology

The development of our knowledge about the DNA sequences brought a large amount
of functional data about biolocial processes. In order to read the amount of information
we need, it was necessary to study formal statistical and computational methods, for
example, when comparing two big DNA strands or building complex phylogenetic trees.
These issues are turning more and more easy to work with thanks to the development of some algorithms that process data more efficiently, being today accesible for
almost everyone even through online softwares.

Objectives

The following project aims the study of the biodiversity of a lake located at the
south of Minas Gerais, Brazil, through Molecular Biology techniques.

3
3.1

Materials and Methods

gDNA Extraction

At first, it was collected materials at a lake and brought to Sao Paulo, Brazil. Before
starting the extration itself the sample was observed through an optcial microscope.
The protocol involved the use of glass beads at a orbital shaker promoting cell lysis
through mechanical breakage, but it was not necessary since the sample used was at
a liquid phase. Another change necessary due to the use of liquids was interrupting
the protocol when the isopropanol was inserted and then resuspending, because the
"cleaning" proposed for soil samples by the protocol would cause losses to the liquid
sample.
In the absence of a cold centrifugue, the centrifugation was made at room temperature. Besides that, the protocol was followed diluting, incubating and centrifugating
so that there was at the end a considerable amount of DNA extracted.
After the extraction, the sample passed through a qualitative analysis with agarose
gel and a quantitative analysis at the spectrophotometer evaluating the absorbance at
different wavelenghts to recognize the purity of the DNA extracted.

3.2

Polymerase Chain Reaction - PCR

It was possible to amplify the ribosomal DNA fragment separately of the DNA
extracted from the organisms of each domain due to the property mentioned before.
For eukarya, the primers used were EUK F/ EUK R, for bacteria they were 27 F/1492
R and for archea arch 21 F/1492 R. The mix was added and the samples were submitted
to the PCR cycling.
After the reaction, the instructors verified the amplicon on agarose gels.

3.3

Insert and Vector ligation and Heat Shock at E. coli

The procedure was followed accordingly to the protocol. The sequence amplified
was inserted at the plasmidi vector pGEM, a linearized strand ending with a T on each
side. This fact increased the efficiency because the polymerase used inserted an A on
each side of the insert.
The vector had already two important regions. One was resistant to ampicilin
and another was a reporter gene with -galactosidase. The resistence to ampiciline
increased the concentration of the strain of interest and the reporter gene garanteed a
different color because the inserted split the genes of the subunit and the subunit
of the galactosidase, preventing the sugar to be reduced. The samples that had a blue
color had the reduction of the sugar so those samples did not receive the insert.
Proceeding, to integrate the plasmidium at a E. coli I utilized the Heat Shock
technique, in which the membrane of the bacteria is thermally permeabilized. The
instructors also integrated the plasmidium using eletroporation.

3.4

Plasmid DNA Extration (Miniprep)

I left the colonies with the instructors to be incubated overnight and after that the
instructors separated the colonies that would be used to proceed with the Miniprep
protocol. To extract the plasmid of interest the vector splited from the rest of the
bacteria with centrifugation.

3.5

Bioinformatic analysis

The Minipreps were sequenciated by the instructors and the data was released as .abi
to be analyzed and then converted into .fasta, a more universal format. With the fasta,
the vector was removed with vecscreen and the resulting sequence was blasted, both
with online NCBI tools[6] . Finally, I made a phylogeny tree to analyze the biodiversity
of the system.

Results and Discussion

Combining the blast result with the phylonegeny tree obtained, we had the following
information:

Figure 1: Phylogenetics Tree with the name of the species

It is important to know that the Sterkiella is also called Oxytricha. The presence
of paramecium was expected and all three species found are plausible, because they all
inhabit in freshwater[6][7] .
The optical microscope exhibited some diatoms and some plants, but neither one nor
the other were found at the sequences. At first, it is possible to say that it happened
because of the cell wall that both plants and diatoms have - diatoms has a silica
deposition in it[8] . However, the work presented some failures that contributed to the
lack of some results.

4.1

Environmental differences

The protocol emphasized soil samples yet the sample I studied was liquid. As
commented at the methods, a few steps were modified in order to overcome the situation, but the reactants and its concentrantions were essentialy the same as ordered the
protocol, what reduced the efficiency of the process.
As shown in the literature[9] , we indeed followed wrong steps, for example the
fixation of the sample with alcohol before extration, what caused a reduced yield as
well as diversity. An example of extraction kit recommended for freshwater samples is
the QiagenDNeasy Blood and Tissue kit (QBT).

4.2

Domain found

It is easy to see at the PCR results that our samples contained Bacterias but not
Eukaryas.

Figure 2: PCR results of Eukarya and Bacteria, respectively

However, the three species are from the Eukarya domain. Initially, a possible
explanation was that it ocurred a misidentification after the PCR reaction or after
the DNA has been sequenced, interchanging the datas. However, I searched for the
primers sequences and blasted with the sequence of Chilomona paramecium. I found
this similarities using the primer 27 F and for the 1492 R, used in bacterias (the query
is the eukarya found and the subject is the primer):

Figure 3: Comparision of the bacteria primers and one Eukarya found

Both primers are 20 bases-length, so it is easy to see that the similarity of 15 bases
found with the 1492 R primer is a reliable way to justify the issue.

Conclusions

The objective was at least slightly attended: there are Sterkiella nova, Paramecium
multimicronucleatum and Chilomonas paramecium at the studied lake and these are
verosimilar results. However, it is important to make changes at the protocol when
manipulating freshwater samples so that a more precise study can be made about the
biodiversity of the environment.

References

[1] ALBERTS, B.; DENNIS, B.; The Cell, Garland Science; 5th edition, 2007
[2] MANHESA, G.; ALLGRE, C. J.; Lead isotope study of basic-ultrabasic layered
complexes: Speculations about the age of the earth and primitive mantle characteristics. Earth and Planetary Science Letters, 1980.
[3] WOESE, C.; KANDLER O.; WHEELIS, M.; Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya, Proc Natl Acad
Sci USA, 1990.
[4] ISENBARGER, T. A.; CARR, C. E.; The Most Conserved Genome Segments for
Life Detection on Earth and Other Planets. Springer Science, 2008.
[5] Max Planck Institute; Department Computational Molecular Biology, available at
<http://www.molgen.mpg.de/en/bioinformatics>, accessed at 07/01/2015.
[6] National Center for Biotechnology Information
available at <http://www.ncbi.nlm.nih.gov/>, accessed at 07/02/2015
[7] DESHMUCK N. Z.; NIKAM S. V.; JAWALE C.S.; SHAIKH T. T.; MORE B.
V.; A new species Oxytricha susheelum from freshwater in Aurangabad, Trends in
Parasitology Research, 2012.
[8] MEDLIN, L. K.; KACZMARSKA, I.; Evolution of the diatoms: V. Morphological
and cytological support for the major clades and a taxonomic revision, Phycologia,
2004
[9] ELAND L. E.; DAVENPORT R.; MOTA C. R.; Evaluation of DNA extraction
methods for freshwater eukaryotic microalgae, Water Research, 2012