1. Molecular approach to environmental
transposon-host interactions
By Dmitrij Kalacov
Supervisor: Dr James McClellan

2. Contents
Abstract......................................................................................................................... 3
Introduction................................................................................................................... 4
Materials & Methods................................................................................................... 10
Approach.................................................................................................................. 10
Sampling.................................................................................................................. 12
DNA extraction: Baffins Pond & Frog Lake .................................................................. 12
DNA extraction: English Channel................................................................................ 14
PCR: primers design and components......................................................................... 15
PCR: amplification of rRNA 16S gene fragments.......................................................... 16
PCR: amplification of transposase genes and sequences neighbouring transposase ...... 17
PCR: junctions amplification ...................................................................................... 18
PCR: purification and precipitation............................................................................. 19
Cloning..................................................................................................................... 20
Plasmids isolation ..................................................................................................... 21
Plasmids purification................................................................................................. 23
Results......................................................................................................................... 24
Samples.................................................................................................................... 24
DNA ......................................................................................................................... 24
PCR.......................................................................................................................... 24
Cloning..................................................................................................................... 28
Plasmids isolation & purification................................................................................ 29
Sequencing............................................................................................................... 30
Discussion.................................................................................................................... 33
Findings.................................................................................................................... 33
Salinity ..................................................................................................................... 33
DNA ......................................................................................................................... 34
PCR.......................................................................................................................... 35
Cloning..................................................................................................................... 36
Plasmids isolation and purification............................................................................. 37
Final results and conclusion....................................................................................... 38
Acknowledgements...................................................................................................... 42
References................................................................................................................... 43

3. 3
Abstract
The diversity in microorganisms is challenging to study: they are difficult to isolate
and characterise. It is widely considered that 90% of bacterial organisms are
uncharacterised on the ground that they are currently unculturable. A derivation of
these facts is that we hardly understand life on Earth at all. It is likely that if it was
possible to access the “vault” of genetic information of currently unculturable
microorganisms, our knowledge of biology would undergo a radical change. The
research aims to derive a method for extraction of genomic information from
environmental samples by exploiting transposon-host interactions. This was
achieved by using consequent steps of a proposed method: [1] cut environmental
DNA with a suitable restriction enzyme; [2] ligate under dilute conditions; [3] PCR
with "out" primers; [4] clone and sequence products. It was vindicated that the
proposed method is capable of capturing and accumulating partial information of
Sulfolobus islandicus YG.57.14 transposase gene from the environmental sample
with no need for culture, albeit failed to retrieve a full transposon sequence of the
organism. Hence, further improvements [e.g. sampling, sets of primers, PCR
conditions calibration] would be beneficial for the proposed method technique.
Additionally, observations provided a collateral background for the ecological aspect
of the study, specifically indicating higher abundance and distribution levels of
transposable genes in the marine environment in contrast to brackish and fresh
aquatic environments. Therefore, the method is applicable and may be of great use
after eradication of pitfalls. It is probable this study may lead to an establishment of
a new robust technique for characterising any aquatic prokaryotic organism.

4. 4
Introduction
Life in general, including aquatic life, is characterised by diversity (Nevo et al. 1984).
Large differences between organisms are the basis of speciation, whilst smaller ones
within a species reflect polymorphisms of various sorts. While some biological
diversity arises from exposure to different environmental conditions [including
epigenetics], it is generally believed that most diversity reflects differences in DNA
sequence (Nevo et al. 1984).
Biological diversity is interesting for several reasons, ranging from fundamental,
through practical, to applied. Fundamentally, without diversity there could not be
evolution by natural selection, since there would be no differences upon which
selection could be based(Van Doorn et al.2009). Practically, diversity gives biological
systems significant buffers against environmental vicissitudes; one example being
variation in resistance to parasites, which is the reason why monocultures are more
vulnerable to disease than more diverse populations (Svensson & Raberg 2010). In
applied biology, variation is of interest because it means there are possibilities to
isolate useful compounds, such as antibiotics and industrially-useful enzymes, given
a good enough understanding of what is available in the environment.
Diversity is relatively easy to study in macroorganisms because each individual
macroorganism is easy to distinguish from each other and each contains relatively-
many copies of its characteristic DNA sequences (Andrews 2012). By contrast,
studying diversity in microorganisms is much harder, because they are difficult to
isolate, and each contains relatively little of its characteristic DNA (Andrews 2012).
Historically microorganisms have been characterised after artificial culture, except

5. 5
for rare cases where some "natural" culture has occurred (Moyer 1929, Greene
2002). Culture requires a good knowledge of the organism's requirements for growth
and scrupulous measures to avoid contamination by parasites, competitors etc.
(Greene 2002). For practical purposes it also requires microorganisms to be capable
of living in an environment tolerable to human beings - for example, we know little
of the conjectured "deep hot biosphere" because scientists cannot live under those
sorts of conditions (L’Haridon et al. 1995, Bell & Heuer 2012, Slobodkin & Slobodkina
2014).
The upshot of these considerations is that rather little is known about bacterial
diversity. Though for obvious reasons exact figures are hard to give, it is widely
thought that 90% of microbial genomes are quite, or completely, unknown
(Kellenberger 2001, Sharma et al. 2005). This ignorance can lead to serious error. For
example, when the Deepwater Horizon rig exploded, the resulting enormous oil spill,
and its projected consequences, lead to serious proposals to seal the well by
dropping a nuclear bomb on it (Quinn 2010). As it turned out, however, before this
scheme could come to fruition, naturally-occurring bacteria, whose existence was
almost unsuspected, had largely bioremediated the problem (Baelum et al. 2012,
Guttierez et al. 2013). Additionally, it is undoubtedly the case that solutions to the
current problem of evolving antibiotic resistance exist in natural organisms - but we
do not know where or what they are, nor how to culture them (Davies & Davies
2010).
In principle the problem is cybernetic. All the required information [genomes of
unculturable organisms] is out there in the environment. Furthermore, it is in some

6. 6
sense meant to be understood - decodable by polmerases and ribosomes. Could
there be a cryptological solution to this cybernetic problem?
This thesis tests a specific cryptological approach to the problem of understanding
the genomes of unculturable organisms. In principle the approach taken is not
dissimilar to that adopted by the WW2 codebreakers at Bletchley Park - a key insight
was a repetitive phrase likely to occur in German messages (Kahn 1980). The
proposed approach also uses a repeated "phrase", likely to occur in all genomes, as
a "crib" to crack the unculturable code.
All cellular organisms are infested by genetic parasites known as "jumping genes" or
"transposons" (Rebollo et al. 2010). All transposons are capable of removing
themselves from their existing host site, in which they constitute an integral, but
foreign, part of the DNA, and reinserting in another site (Rebollo et al. 2010). Some
transposons in eukaryotes are also capable of producing an RNA copy that can be
converted back into DNA: these are called retroposons, and HIV is an example (Chow
et al. 2009).
The mechanism of transposition is quite well worked out, and relies upon an enzyme
calledtransposase (Ivancevic et al. 2013, Wiemann et al. 2013). This enzyme attaches
sequence-specifically to the ends of transposons where it makes specific cuts, and
also non-specifically to a target site elsewhere in the genome [in actual fact there
may be some specificityto target sitechoice, but there are always very many possible
targets in the host genome] (Levin & Moran 2011). Then transposase rejoins the
transposon ends to the ends in the new target site, resulting in a transposition of the
element from one site to another (Levin & Moran 2011). Variations on this process

7. 7
can include leaving a copy in the original site, but often the effect on the original site
is to leave a double-stranded break, as Barbara McClintock famously deduced in the
course of the work that won her the Nobel Prize for discovering transposons (Pray &
Zhaurova 2008).
Transposons are very successful genetic elements, so much that transposase genes
constitute the largest group of genes in known genomes (Aziz et al. 2010, Leclercq &
Cordaux 2011). Almost if not actually every organism ever studied has its own
transposons, albeit the host range of an individual transposon is likely to be fairly
limited: where it is not, opportunities for horizontal gene transfer may be increased
(Levin & Moran, 2011).
Although transposons vary from organism to organism [and within organisms]
transposase itself exhibits a high degree of sequence conservation, doubtless
reflecting the factthat the basicchemistry of transposition is the same: tyrosine side-
chains making transient covalent bonds with DNA, and then DNA chains reforming
by nucleophilic attack of 3'OH groups on the tyrosine-DNA ester (Levin & Moran,
2011). A particular region of the transposase gene shows two areas of high
conservation thus identifying sequences that are common, and repeated, in all
cellular organisms.
This transposase conservation represents a "crib", and the strategy adopted to
exploit it in unravelling the genomes of unculturable organisms was as follows. First,
DNA was prepared from a heterogeneous collection of environmental
microorganisms, obtained from three aquatic sites in or near Portsmouth, England.
PCR primers were designedto amplify the conserved region of transposase DNA. PCR

8. 8
with these primers would give an idea of the presence of transposase sequences in
the environmental sample. The complements of these primers, designated "out",
would amplify nothing at all in principle, except in cases where the transposon
resided on a small circle. However, the approach was to use these "out" primers in
pairwise combinations with established primers that amplify known and highly-
conserved functional genes, namely those encoding 16S ribosomal RNA (Pei et al.
2010, DePristo et al. 2011, Barquist et al. 2013). In principle, this will give PCR
products in the rare cases where a transposon has settled down close to a ribosomal
RNA gene. If it succeeds, sequencing the PCR product should reveal several pieces of
crucial information, namely [1] the identity of the host clade, from the sequence of
the rRNA gene; [2] the actual sequence of the transposon that infests it; [3] the fact
that this transposon, and that host, are connected.
The primary research question in this thesis is whether or not such a scheme has any
chance of success, but it was obvious at the outset that should it work, future
possibilities would be immense. For example, if one can find a natural transposon-
host junction it is then in principle possible to sequence the whole genome of that
organism, without the requirement for culture. This would be done as follows: [1] cut
environmental DNA with a suitable restriction enzyme [it mustn't cut between the
two "out" primer-binding sites]; [2] ligate under dilute conditions, so as to make
circles; [3] PCR with "out" primers; [4] clone and sequence products. Each product
will give the context of the transposon from a particular site, and one will be able to
tell when the left-hand-side context ends and the right-hand-side context begins,
because the chosen restriction site will mark it.

9. 9
In addition to the primary research question, broadly the usability of transposase as
a "crib" for difficult genomes, a secondary research question related to the
distribution of transposons in different environments. To address this, samples of
aquatic microorganisms were collected from three sites: [1] open sea [the English
Channel], [2] brackish water [Frog Lake, to the East of Portsmouth island], [3] fresh
water [Baffins Pond]. Time did not allow a properly-systematic approach to this
question, but any observed differences could form the basis ofimproved insights into
transposon ecology.

10. 10
Materials & Methods
Approach
The main approach was to isolate and characterise naturally-occurring transposon-
host junctions from environmental samples. This was achievedvia useof transposons
and transposase natural molecular features and application of these as jumping
“tags” [Figure 1, p. 11]. In the event of success, it was proposed to use tn tagged
sequences for genome deciphering [Figure 2, p. 11]. The main method involved PCR
using primers that read into transposase [positive control], and for the experiment
itself, their complements [reading out], were used in pairwise combinations with
either one or the other of primer pairs already established to amplify rRNA genes
from prokaryotes (Mori et al., 2013). A supplementary method involved using only
primers for rRNA genes but greatly expanding the incubation time. This allowed the
isolation of large clones that were expected to contain a transposon inserted into an
rRNA gene.
The experiments were repeated with longer extension times and higher nucleotide
concentrations, with the aimof generating long fragments that would result from the
insertion of transposons into rRNA. Such events would of course often be lethal, but
not always, since rRNA genes may be duplicated, and in any event environmental
DNA will also include that from moribund organisms, provided it is not too heavily
degraded (Fedoroff 2012). The second approach was to take primers that amplify
sections of transposon DNA, based on the sequence conservation exhibited by
transposasegenes. Application of these primers to environmental DNA was assumed
to amplify all transposons present.

11. 11
Figure 1: How transposons(TN) jumping tag sequences
Figure 2: Use of TN-specificprimers fortagging sequencesand
subsequentgenomedeciphering (knownTN sequence[red];
unknown HOSTsequence[blue])

12. 12
Sampling
Three distinct sampling sites were chosen based on a conjectural salinity of water to
cover a wider range of aquatic habitats – fresh water [Baffins Pond], brackish water
[Frog Lake] and sea water [English Channel]. Fresh and brackish water samples were
collected in sterile 1000 ml glass containers, 5 – 10 cm below the water surface and
sealed to avoid contamination (Al-Harbi & Uddin 2005). Sea samples were provided
by University as a part of BSc practical. Next, 100 ml from each sample were filtered
through 0.22 µm filters by pump-filtering (Hammes et al. 2010). Water salinity was
measured using conductivity meter. Filters with filtrate were taken for the DNA
extraction; leftovers of water samples were stored in a cold room at average
temperature of 4o C.For subsequent post-extraction PCR procedures, the fresh water
sample was marked as B, brackish water sample was marked as F, the sea water
sample was marked as S, and the control no template marked as C.
DNA extraction: Baffins Pond & Frog Lake
The coloured filtrate was scraped off each filter into separate 1.5 ml microcentrifuge
tubes using a sterile scalpel for subsequent DNA extraction using Qiagen DNeasy
Blood & Tissue Kit (Fuhrman et al. 2008). The DNA extraction kit was chosen because
it proved to be more convenient for a high quality sample processing (Polley et al.
2011). The DNA extraction was conducted following the classicsuccessiveprocedure:
[1] lysis, breakdown of a cell; [2] capture & cleaning, removal of all unnecessary
substances and [3] elution of DNA, production of a high quality DNA (Urakawa et al.
2010). The direct procedure:

13. 13
[1] Lysis. Added 180 µl of buffer ATL and 20 µl of Proteinase K to microcentrifuge
tubes containing scraped off filtrate and incubated at 56o C for 1 hour.
[2] Capture & Cleaning. Post-lysis mixture vortexed for 15 seconds. Added 20 µl of
Buffer AL and vortexed for 15 seconds. Subsequently, added 200 µl of absolute
ethanol to the mix and vortexed for 15 seconds. Results mixture was transferred to
DNeasy Mini Spin column within a 2 ml collection tube by pipetting and centrifuged
for 1 minute at maximum speed (*10.000 rpm). DNeasyMini Spin column transferred
to a fresh 2 ml collection tube. Added 500 µl of Buffer AW1 and centrifuged for 1
minute at maximum speed*. DNeasy Mini Spin column transferred to a fresh 2 ml
collection tube. Added 500 µl of Buffer AW2 and centrifuged for 3 minutes at
maximum speed* to dry the DNeasy membrane and avoid contamination with
ethanol.
[3] Elution. Moved DNeasy Mini Spin column to a fresh 1.5 ml centrifuge tube. Added
100 µl of Buffer AE by pipetting directly onto DNeasy membrane. The mixture was
incubated at the room temperature for 1 minute and subsequently centrifuged for 1
minute at the maximum speed*. Elution step was repeated to increase yield.
Samples were tested by a NanoDrop spectography and agarose gel electrophoresis
to approve presence of significant amounts of DNA. Exact gel contents: 0.4 of
agarose powder in 40 ml of TBE buffer [per 100 ml: tris-HCl = 12.11 g, boric acid =
5.56 g, EDTA = 0.37 g]. Load: 4 µl of dye [visualisation] + 20 µl of DNA; marker = 1
kbp. Agarose gel conditions: 100 volts for 1.5h. After the run, gels were placed on a
tray with buffer. Added 50 µl of ethidium bromide and incubated at a room

14. 14
temperature for 10 minutes to visualise the DNA under UV light. Afterwards, DNA
was preserved in a freezer at a temperature of -20o C (Fierer et al. 2010).
DNA extraction: English Channel
The sea sample was extracted during a practical using PowerBead method and DNA
extraction protocol [below] (Salcher et al. 2011). The DNA extraction was conducted
following the classic successive procedure: [1] lysis, breakdown of a cell; [2] capture
& cleaning,removal of allunnecessary substances and [3] elution of DNA, production
of a high quality DNA (Urakawa et al. 2010)The most promising samples were taken
for subsequent processes. The direct procedure:
[1] Lysis. Added 0.25 grams of the sample to the Powerbead Tube and briefly
vortexed. Added 60 µl of Solution C1 and vortexed for 10 minutes. Centrifuged the
mixture for 30 seconds at maximum speed [*10.000 rpm] (Willner et al. 2012).
[2] Capture & Cleaning. Transferred 500 µl of supernatant to a fresh 2 ml collection
tube. Added 250 µl of Solution C2 and vortexed for 5 seconds. Incubated the mixture
at 4o C for 5 minutes and centrifuged at a room temperature for 1 minute at
maximum speed*. Transferred 600 µl of supernatant to a fresh 2 ml collection tube
without disturbing the pellet. Added 200 µl of Solution C3, briefly vortexed and
incubated at 4o C for 5 minutes. Further, centrifuged the mixture at room
temperature for 1 minute at maximum speed*. Pipetted 750 µl of supernatant into
a 2 ml collection tube without disturbing the pellet.

15. 15
[3] Elution. Added 1200 µl of Solution C4 and briefly vortexed. Loaded 675 µl of the
mixture directly onto a spin filter and centrifuged for 1 minute at maximum speed*.
Discarded the flow through and added extra 675 µlof supernatant onto the spin filter
and centrifuged for 1minute atmaximum speed*. Loaded the remaining supernatant
onto the spin column and repeated the previous step. Added 500 µl of Solution C5
and centrifuged the mixture for 1 minute at maximum speed*. Discarded the flow
through and repeated previous centrifuging step. Carefully placed the spin filter in a
clean 2 ml collection tube, added 100 µl of a molecular grade water and centrifuged
for 1 minute at maximum speed*. Subsequently, discarded the spin filter and
checked the result DNA on a NanoDrop spectographer. DNA was stored in a freezer
at a temperature of -20o C for preservation. Tested via electrophoresis.
PCR: primers design and components
The rRNA 16S gene fragments, transposase genes and sequences neighbouring
transposase were amplified with primers via PCR. Tn primers used for PCR were
designed based on a strong consensus between protein sequences (Kulkosky et al.
1992). Primers included [sequences]: 5’-CAYCAYACNGAYANNGGNNNNCARTA-3’
[primer 1, reverse in]; 3’-GTRGTRTGNCTRTNNCCNNNNGTYAT-5’ [primer 2, forward
out]; 5’-AAYGGNATGATHGARNNNCAYGGNNNNNTNAA-3’ [primer 3, reverse out]; 3’-
TTRCCNTACTADCTYNNNGTRCCNNNNNANTT-5’ [primer 4, forward in]. RNA primers
included standard 8-27F [5’-AGAGTTTGATCMTGGCTCAG-3’] and 1512-1492R [5’-
ACGGYTACCTTGTTACGACTT-3’].

16. 16
PCR required a specific set up for each amplification [rRNA, transposase, sequences
neighbouring transposase and junction amplifications]. The list included molecular
grade water and the master mix. The Master Mix contained: 10xbuffer = 2.6 µl,
enzyme = 1 µl and dNTPs = 2.6 µl [per reaction] (Nadkarni et al. 2002).
PCR: amplification of rRNA 16S gene fragments
The rRNA 16S gene fragments were amplified via PCR using specific primers [8-27F
and 1512-1492R] under two different conditions (Schloss et al. 2011). The conditions
list included PCR using “rRNA” conditions and PCR using “transposase” conditions
[Table 1, p. 17].
Samples under “rRNA” conditions were incubated for 3 minutes at 94o C [hot start]
and denatured for 45 seconds at 94o C prior annealing. Subsequently, samples were
annealed for 1 minutes at 57o C and polymerisedfor 2 minutes at 72o C. The entire
PCR process was repeated for 40 cycles and finished off with the final extension for
7 minutes at 72o C.
Samples under “transposase” conditions were incubated for 3 minutes at 94o C,
denatured for 45 seconds at 94o C, annealed for 2 minutes at 50o C and polymerised
for 30 seconds at 55o C. The entire cycle was repeated 40 times and ended with the
final extension for 7 minutes at 72o C. PCR result products were tested via gel
electrophoresis.

17. 17
Table 1: PCRamplification of rRNA 16S gene fragmentsconductedundertwo conditions.
PCR contentsproportionsforeach tuberepresented in µl. Each tubecontained 25 µl of a
mixture
Baffins Frog Sea No Template
Baffins DNA 0.6 - - -
Frog DNA - 1.8 - -
Sea DNA - - 3.3 -
GDW 17.2 16.0 14.5 17.8
Master Mix 6.2 6.2 6.2 6.2
8-27F Primer 0.5 0.5 0.5 0.5
1512-1492R Primer 0.5 0.5 0.5 0.5
PCR: amplification of transposase genes and sequences neighbouring
transposase
The PCR amplification of transposase genes was conducted using reverse in and
forward in primers [Table 2, p. 18]. The PCR process included a hot start for 3 minutes
at 94o C and continued with 40 cycles of denaturation for 45 seconds at 94o C,
annealing for 2 minutes at 50o C and polymerisation for 30 seconds at 55o C. Final
extension lasted for 7 minutes at 72o C.
The PCR amplification of sequences neighbouring transposase included application
of reverse out and forward out primers [Table 3, p. 18]. The PCR procedure included
a hot start for 3 minutes at 54oC and a final extension for 7 minutes at 72o C. In-
between, 40 cycles of denaturation [45 seconds at 94o C], annealing [2 minutes at
50o C] and polymerisation [130 seconds at 55o C] were performed.

18. 18
Table 2: PCRamplification of transposasegenescontentproportionsforeach tube
represented in µl. Each tubecontained 25 µl of a mixture.
Baffins Frog Sea No Template
Baffins DNA 0.6 - - -
Frog DNA - 1.8 - -
Sea DNA - - 3.3 -
GDW 17.2 16.0 14.5 17.8
Master Mix 6.2 6.2 6.2 6.2
Reverse In 0.5 0.5 0.5 0.5
Forward In 0.5 0.5 0.5 0.5
Table 3: PCRamplification of sequencesneighbouring transposasecontentproportionsfor
each tuberepresented in µl. Each tubecontained 25 µl of a mixture.
Frog Sea No Template
Frog DNA 1.8 - -
Sea DNA - 3.3 -
GDW 16.0 14.5 17.8
Master Mix 6.2 6.2 6.2
Reverse Out 0.5 0.5 0.5
Forward Out 0.5 0.5 0.5
PCR: junctions amplification
The purpose of junction amplification was production of viable genetic material
capable of fulfilling plasmids isolation. Therefore, it was decided to conduct and
simulate every possible occasion prior the NanoDrop spectography and gel
electrophoresis. The finalPCR run included a wide range of DNA samples and primers

19. 19
to amplify all possible combinations of DNA with rRNA forward/reverse primers and
forward/reverse tn primers [Table 4, p. 19].
Table 4: PCRjunction amplification contentproportionsforeach tuberepresented in µl.
Each tubecontained 25 µl of a mixture.
Frog
1
Frog 2 Frog 3 Frog 4 Sea 1 Sea 2 Sea 3 Sea
4
Frog DNA 1.8 1.8 1.8 1.8
Sea DNA 3.3 3.3 3.3 3.3
GDW 16 16 16 16 16 16 16 16
8-27F Primer 0.5 0.5 - - 0.5 0.5 - -
1512-1492R
Primer
- - 0.5 0.5 - - 0.5 0.5
Forward Out 0.5 - 0.5 - 0.5 - 0.5 -
Reverse Out - 0.5 - 0.5 - 0.5 - 0.5
Master Mix 6.2 6.2 6.2 6.2 6.2 6.2 6.2 6.2
PCR: purification and precipitation
[1] Purification. All PCR products were purified and precipitated to improve the
quality of DNA. The following instructions were applied to every PCR reaction.
First, 110 µl of Buffer PB were added to the remainder of the PCR reaction, mixed
and pipetted into the QiaQuickMini spin column placed in a 2 ml collection tube. The
mixture was centrifuged for 1 minute at the maximum speed [*10.000 rpm]. The
flow-through was discarded. Added 750 µl of Buffer PE and centrifuged for 1 minute
at maximum speed*. Discarded the flow-through and centrifuged once more under
the same conditions. Transferred QiaQuick Mini spin column to a clean 1.5ml
microcentrifuge tube and pipetted 30 µl of Buffer EB directly onto the QiaQuick

20. 20
membrane. Incubated at room temperature for 1 minute and centrifuged under the
same conditions: 1 minute at maximum speed* (Fuhrman et al. 2008).
[2] Precipitation. The following mixture was prepared according to the protocol: 22
µl of DNA + 2 µl of 4M NH4Az. The mixture was stored in the freezer at -20o C for 10
minutes. Subsequently, the mixture was spun for 10 minutes at the maximum speed.
Removed the supernatant. Afterwards, added 200 µl of fresh 70% ethanol [170 ml of
ethanol + 30 ml of distilled water]. The mixture spun at maximum speed* for 10
minutes. Pellet formed, therefore supernatant was removed and pellet dried at a
room temperature for 30 minutes. Added 30 µl of molecular grade water. The result
mixture was analysed via the NanoDrop spectography to confirm a sufficient quality
of samples.
Cloning
To prepare for the cloning transformation, a water bath was warmed up to 42o C and
SOC medium was warmed up to room temperature. Prepared LB plates [200 ml of
water, 4 g of LB Lennox broth powder and 3g of 1.5% agar bacteriological;autoclaved
at 121o C for 15 minutes] with 100 µg/ml of ampicillin by spreading 100 µl of x-gal
and incubating at 37o C. One vial of competent cells stored on ice prior each
transformation (Yasui et al. 2009).
[1] The set up. The Frog Lake cloning reaction required 2.4 µl of DNA, 1 µl of salt
solution, 1 µl of TOPO vector and 1.6 µl of a molecular grade water. The sea sample
cloning reaction required 2.2 µl of DNA, 1 µl of salt solution, 1 µl of TOPO vector and

21. 21
1.8 µl of a molecular grade water. Contents were gently mixed by tapping the side of
the tube and incubated at a room temperature for 30 minutes (Yasui et al. 2009).
[2] Transformation. Added 2 µl of the cloning reaction to each vial of thawed
competent cells and mixed gently by tapping the side of the tube. Incubated on ice
for 5 minutes, heat-shocked cells by placing transformation tube into a bath
containing 42o C water for 30 seconds and subsequently transferred back on ice.
Added 250 µl of SOC medium to each tube. Incubated tubes at 37o C in the orbital
incubator with a shaking set up for 200 rpm for 1h. Later, spread 150 µl of each
transformation on a pre-warmed LB plate. After, plates were transferred to an
incubator (37o C) for overnight incubation (Chung et al. 1989).
[3] Streak. Single colonies were picked using sterile loops [sterilised using ethanol
and burner] and streaked around fresh LB plates (Davis and DiRita 2008). Both blue
and white colonies streaks were made using S1-3, S2-4, F1-2 and F3-4 samples.Streak
subjects were marked according to the identification code: Site-Sample-Colony-
Origins. For example, S4W0 (S for Sea, 4 for 2-4 sample [Table 4, p. 17], W for White
and O for Original plate). Subsequently, plates were relocated back to incubator for
overnight.
Plasmids isolation
[1] Preparation. The media was prepared prior DNA incubation in universals.
Universals media contained 4 g of Lennox broth powder per 200 ml of distilledwater.
The media was autoclaved and mixed with 1 ml of ampicillin. Next, media was
pipetted into universals, 5 ml each (5 x 20) - in overall 20 universals were made in

22. 22
response to distribution and quantity of perfectly single colonies on the plates: S2-4
white x 2, S1-3 white x 2, F3-4 blue x 2, S2-4 blue x 2, S2-4 white x 2, S2-4 blue original
x 2, F2-4 blue original x 2, F2-4 white originalx 2, S2-4 white original x 2, Control white
x 2.
Single streak colonies were moved to universals and mixed. The direct procedure
included dipping a loop into ethanol, burning [sterilisation], picking up single colony,
burning edges of a universal [sterilisation], mixing, burning edges [sterilisation],
sealing a universal and sterilising the loop before repeating the procedure. Finally,
universals were placed in a shaking incubator overnight at 200 rpm.
[2] Isolation. Added 2 ml of overnight reaction to 2 ml bullet tube and spanned for 1
minute at maximum speed. Successfulreactions formed pellets.The supernatant was
discarded and tubes were dried with a tissue without touching the pellet on the
bottom of the tube. Resuspended the pellet in a 100 µl of resuspension Solution I
[EDTA = 2.92 g/L, TrisCl = 3.95 g/L, glucose = 9.01 g/L] and vortexed briefly. Added
200 µl of a Solution II [2M NaOh = 1ml, 10% SDS – 1ml, molecular grade water – 8ml]
and mixed gently. Added 150 µl of an ice cold Solution III [100 ml 5 KAc + glacial
acetic acid 19.2ml + molecular grade water 47.6ml] and stored on ice for 30 minutes.
Later, centrifuged for 10 minutes at maximum speed [*10.000 rpm]. The supernatant
was transferred to a fresh tube, the old tube containing white sediment got
discarded. Added 1ml of an absolute ethanol and centrifuged for 10 minutes at
maximum speed*. Poured out the supernatant, added 1ml of fresh 70% ethanol and
centrifuged for 5 minutes at maximum speed*. Poured out the supernatant and tried

23. 23
the tube with a tissuewithout touching the pellet. Resuspended in 20 µlof molecular
grade water prior gel electrophoresis.
[3] Gel electrophoresis. Wells were loaded with 5.5 µl of dye and DNA mix (1 µl dye
+ 5 µl DNA). Used 1 kbp marker as a scale. Gel electrophoresis run lasted for 3h at
200 V to spread potential plasmid bands across the gel. Before UV light test, gels
were stained by 45 µl of ethidium bromide for 15 minutes.
Plasmids purification
The DNA was transferred to a spin column and centrifuged for 1 minute at the
maximum speed*. The flow-through was discarded and 0.5 ml of a buffer PB were
added to the tube. Centrifuged the tube for 1 minute at maximum speed and
discarded the flow-through. Added 0.75 ml of a buffer PE to the column and
centrifuged for one minute at maximum speed. Discarded the flow-through and
centrifuged once more to get rid of the residual wash buffer. Transferred the column
to a fresh tube, added 50 µl of a buffer EB (elution), incubated at room temperature
for 1 minute and centrifuged for 1 minute at maximum speed*. Checked the result
DNA via the NanoDrop spectographer to validate presence of sequencable plasmids
(Sharan et al. 2009). After confirmation, genetic material was sent off to Eurofins
Genomics for sequencing.

24. 24
Results
Samples
Water samples test for conductivity and pH results [Table 5, p. 24].
Table 5: conductivityand pHtest results;high levels of conductivity indicatehigher levels of
salinity
Site Conductivity pH
Sea 3999+ (max) 7.91
Baffins Pond 377 7.87
Frog Lake 1300 8.04
DNA
The DNA extraction and purification was successfully performed following the
protocol. As a result of the NanoDrop spectogrpahy, the final set of DNA samples
contained 24.5 ng/µl in the fresh water sample [Baffins Pond], 8.8 ng/µl in the
brackish water sample [Frog Lake] and 4.5 ng/µl in the sea sample [English Channel].
PCR
The agarose gel electrophoresis for PCR of transposase genes revealed strong and
rich genetic material in the sea [S] sample [Figure 3, p. 26] in comparison to other
samples. The PCR for genes neighbouring transposase gel did not show any
significant DNA contents in the sea [S] sample, but revealed a great amount of DNA
in the Baffins [B] pond sample [Figure 4, p. 26]. Among all samples, the Frog Lake
sample contained the weakest traces of DNA.

25. 25
The PCR of rRNA genes was performed under two different conditions – “rRNA” and
“transposase” conditions [Figures 5 & 6, p. 27].
The junctions amplification using 12 different combinations of DNA, primers and sets
of conditions was successful and resulted in an enormous amount of single bands
across different samples [Figures 7 & 8, p. 28].
All PCR products were pooled in groups: Sea 1 and Sea 3 [S1-3], Sea 2 and Sea 4 [S2-
4], Frog Lake 1 and Frog lake 2 [F1-2], Frog Lake 3 and Frog Lake 4 [F3-4]. The
NanoDrop spectography analysis showed following concentration and purity results
[Table 6, p. 25].
Table 6: spectography resultsforpre-cloning PCRjunction amplification samples;260/280
indicatesgenetic material purity – 1.90 is widely considered to be themaximumpurity level
Pool Concentration (ng/µl) 260/280
S1-3 5.1 1.72
S2-4 50.6 1.90
F1-2 37.7 1.87
F3-4 6.8 1.54

26. 26
Figure 3: PCR of transp.genes.The sea
sample reveals a distinctively large
variation of small bands (<0.5 kb) in
comparison to the fresh, brackish and
no template control samples.
Figure 4: PCR for neighbouring
transposase. Strong small bands
(<0.5 kb) in the brackish sample
[Frog Lake]

27. 27
Figure 5: PCR using "rRNA" conditions. All
samplesrevealed potentialtn genescarriers
[~1.5 kb]
Figure 6: PCR using
"transposase" conditions
proved to be less successful
and promising with the setof
weaker bands [~1.5 kb]

28. 28
Cloning
All plates, except the F1-2 plate, contained numerous single colonies. S2-4 original
and F3-4 original plates contained both blue and white isolated singular colonies. As
a result of streaking, S2-4 exposed a vast amount of blue and white colonies, S1-3
exposed white colonies and F3-4 revealed blue colonies. Both original and streaked
colonies were used to make universals before plasmids isolation. All universals
displayed positive signs of growth.
Figure 7: PCR junction
amplification [F1-F4]. F2
and F4 contained rich
genetic material
Figure 8: PCR junction
amplification [S1-4].S2and
S4 samples contained rich
genetic material

29. 29
Plasmids isolation & purification
After plasmids isolation and purification, result DNA and dye mixtures were tested
via agarose gel electrophoresis. Gel scanning revealed multiple large strong bands
(DNA) and weak long streaks (RNA) in three gels. The first gel [Figure 9, p. 29] did not
contain strong single bands. The second gel [Figure 10, p. 29] contained blurred
bands and therefore could not proceed to future procedures and analysis. The third
gel [Figure 11, p. 30] represented both applicable and non-applicable bands.
Figure 9: Plasmids isolation – I.
Multiple bands with no signs of target
plasmids. First letter = site; second
number= sample,third letter = typeof
colony, fourth letter = O indicates
original plates. S = sea; F = brackish; B
= fresh; C = no template control.
Figure 10: Plasmids isolation – II.
Corrupted samples made it hard to
establish if there are any plasmids.
First letter = site; second number =
sample, third letter = type of colony,
fourth letter = O indicates original
plates.S = sea;F = brackish;B = fresh;
C = no template control.

30. 30
Sequencing
Sequencing summary results included both sequence codes and additional
information beneficialfor analysis,suchas the length of the result sequence [e.g.920
nt for S4WO] and the sequence of the primer used for sequencing [M13]. Despite
the wide range of samples, only three were chosen to be sent off for sequencing –
S2-4 [original], S2-4 [streak] and S1-3 [original].
Figure11: Plasmidsisolation – III.Sea
sample contained a plasmid. First
letter = site;second number=sample,
third letter = type of colony, fourth
letter = O indicatesoriginalplates.S =
sea; F = brackish; B = fresh; C = no
template control.

31. 31
All sequences were analysed using the RCSB PDB [Research Collaboratory for
Structural Bioinformatics Protein Data Bank] and the BLAST [Basic Local Alignment
Search Tool] (Altschul et al. 1997). To get to the root of a hypothetical transposon-
host junction, it was required to detect and exclude all unnecessary parts, such as
vectors and M13 primer. Vectors were identified using the BLAST software and the
official map of pCR II-TOPO [used for cloning], including sequences of Hind III, Kpn I,
Sac I, BamH I, Spe I, BstX I, Ecor I, Ecor V, Not I, Xho I, Nsi I, Xba I and Apa I. Later,
rRNA primers [8-27F and 1512-1492R] and tn primers were identified to mark the
ends of the sequence of the interest (Chou & Holmes 2001). To identify
hypothetically present tn sequences, both rRNA and tn primers were highlighted
within a result sequences to be used as pivots for analysis.
The intent analysis of S1-3 sequence detected a degradation of the sample - only one
vector and M13 primer were detected within a sequence. The rest of the sample
sequence was most likely an artefact with no presence of rRNA, tn primers and,
therefore, target genes.
Analysis of S2-4 [original] was successful and included vectors on both sides of the
sequence, M13 primers, rRNA primers and tn primers used for PCR [Figure 12, p. 32].
Sequencing of independently cloned, isolatedand purified S2-4 revealed the identical
sequence to the original S2-4 sample, including vectors and primers.
Short sequences [50 nt] neighbouring tn primers were BLASTed and analysed for
presence of certain genes, including transposase and transposons. BLASTing results
exposed a list of various genes of a similar structure; conversely, only Sulfolobus
genus results contained transposase. According to results and analysis of Sulfolobus

32. 32
transposase and transposon genes database, it was detected that sequence
neighbouring tn primers contains partial sequence of transposase genes, present in
Sulfolobus islandicus Y.G.57.14 [e.g. GeneID YG5714_RS03065: range 588929 –
590167 and GeneID YG5714_RS03445: 660101 – 661345 genes]. In case of
YG5714_RS03445, transposase gene and S2-4 sequence alignment revealed
practically perfect match [sequence of 21 nucleotides long: 5’-
AGGGAAATAGAGGAAGA.TAATC-3’]. Other genes on the list included nor less
interesting results, such as transposase mutator type genes; however, the target
gene [transposase] parts were detected and located only within specific Sulfolobus
organisms, containing 13 – 21 tn long sequences across species of the same genus.
Figure 12: S2-4 sequence[red = vector,blue = M13 primer, yellow = rRNA primer, black= tn
primer, plain = S2-4 DNA,underline= targetareas]

33. 33
Discussion
Findings
 Sampling sites varied in salinity, but revealed equivalent levels of pH
 The fresh water site [Baffins Pond] contained larger amounts of DNA than the
brackish site [Frog Lake] and sea site [English Channel]
 The junctions’ amplification using rRNA forward and transpose out reverse
primers was successful and provided rich bands
 Cloning was successfully performed with the majority of junction
amplification samples [except F1-F2 mixture] which contained a combination
of tn primers, rRNA forward primers and DNA
 Sequencing results revealed partial transposase gene sequences of genus
Sulfolobus within the S4 sample, confirming the efficiency of the proposed
methods despite a number of flaws and a demand for improvement of several
aspects
 The sea sample proved to contain more transposase, and therefore
transposons
Salinity
As expected, salinity levels between the sea, fresh water and brackish water samples
were different. According to the conductivity results, Baffins Pond is the only fresh
aquatic environment with a conductivity level of 377 [fresh water]. Frog Lake’s
conductivity level indicated its brackishness, 1300, which could be due to its
proximity to Langstone Harbour [saline environment with an intense tidal activity],

34. 34
~50 m (Fu et al 2011). The sea water sample’s conductivity showed the maximum
capacity, 3999+. Nevertheless, pH levels of all sampled environments were close to
identical: sea 7.91, fresh 7.87 and brackish 8.04. There could be several explanations
why these distinct environments are so alike in terms of pH. One of the possible
reasons is their relative proximity to each other on a geographical scale within a
highly active and changeable area of the UK.
DNA
The original set of the extracted and purified DNA contained distinctively different
volumes of genetic material. The Baffins Pond sample contained 24.5 ng/µl of DNA,
more than 8.8 ng/µl in the Frog Lake sample and 4.5 ng/µl in the sea sample. Such
findings could be justified by the fact that Baffins Pond is a small-scale pond located
in the middle of a busy residential area of the most densely populated city in the UK
(Maguire et al.2012). Possibly, Baffins Pond might be polluted with both organic and
inorganic substances as a result of an intensive anthropogenic activity, raising
microbial density and nutrients levels. Oppositely, Frog Lake is a brackish
environment located on the edge of the city and surrounded by a dense vegetation
restricting access. The water sample from the Frog Lake was completely transparent
with no signs of major pollution or eutrophication. On the other hand, low DNA
concentrations in the sea sample were surprising due to the fact that the sea water
was expected to be rich in DNA (Caporaso et al. 2012).

35. 35
PCR
The PCR for transposase genes and the PCRfor genes neighbouring transposasewere
conducted as preparation for junctions’ amplification to test primers interaction with
samples. According to the PCR of genes neighbouring transposase, it was detected
that the sea sample contained a large variety of distinct single and multiple DNA
bands in contrast to the no template control, Baffins Pond and Frog Lake samples. In
contrast to the PCR for transposase results, the PCR for genes neighbouring
transposase did not show any signs of genetic material in the sea sample, but
revealed enormous amounts of genetic material in the Baffins Pond sample [did not
contain distinct single bands]. Such results could be supported by the preceding DNA
extraction analysis – the Baffins Pond sample was rich in non-applicable bits of DNA.
Also, EnglishChannel is aconstantly changing environment rich in biotic material and
was expected to deliver positive results.
The PCR for rRNA genes was conducted under two different conditions and resulted
in two distinct sets ofresults. The PCRof rRNA genes conditions was conducted under
original “rRNA” conditions which provided a set of distinct strong single bands of the
same size [assumed to contain target sequences]. The PCR of rRNA genes under
“transposase” conditions revealed minor blurry bands within a different size range
[in comparison to “rRNA” conditions results] which proved effectiveness of “rRNA”
conditions.
The final PCR results, junctions’ amplification, revealed the most efficient
combination of primers for amplification of target genes, transposase and
transposons. The agarose gel electrophoresis visualised the differences between the

36. 36
PCR samples amplified under the same conditions, but with different combinations
of primers. Both brackish and sea results were similar, although the sea sample
clearly revealed a wider range of genetic material across all four conditions [1 – 4].
The F1 and the S1 did not contain any strong distinct bands. The F3 and the S3 did
contain weak bands, but in overall F2, S2, F4 and S4 were considered to be better
options based on a large amount of strong distinct single bands across all different
ranges.
According to the NanoDrop spectography results, the combination of rRNA forward
and transposase out reverse primers revealed a rich DNA concentration [S2-4 = 50.6
ng/µl; F1-2 = 37.7 ng/µl)] with a potential set of target genes. Additionally, S2-4 and
F1-2 demonstrated high levels of purity [260/280] and low levels of contamination
[S2-4 = 1.90; F1-2 = 1.87], since 260/280 = 1.90 is widely considered to be a sign of
pure and non-contaminated sample. S2-4 post-PCR pool alsocontained rRNA reverse
combination and might have had a positive effect on the quality of S2-4 pool,
therefore F3-4 pool was expected to show positive results. However, F3-4 pool purity
levels [260/280] were low [1.54] and might have had an effect on its DNA
concentration [6.8 ng/µl] – it is reasonable to hypothesise that this particular pool
was most likely contaminated. S1-3 pool contamination levels were moderate [1.72],
but DNA concentration was on the edge of its aptness [5.1 ng/µl].
Cloning
Cloning was successfully performed for the majority of plates [except the F1-2 pool
plate]. Possible reason for F1-2 cloning failure could be the poor quality of sample

37. 37
DNA (Chou & Holmes 2001). Both S2-4 and F3-4 plates contained blue and white
single colonies, which indicated a good quality DNA within S4 and F4 samples – such
assumption can be supported by NanoDrop and gel electrophoresis results. S1-3
plate contained a large amount of single white colonies as a result of DNA extraction
and PCR. All plates [excluding F1-2] were used to make universals and were proven
to be successful based on an intense growth signs during incubation.
Plasmids isolation and purification
The purpose of post-isolation-and-purification agarose gel run was to detect single
isolated DNA bands larger than 10 kb - such bands could contain plasmids (Stadler et
al.2004). The first gel[Figure 9, p. 29] contained DNA, but did not result in any strong
singular bands. The second gel [Figure 10, p. 29] could possibly contain plasmids, but
was withdrawn. There are numerous possible causes for genetic material quality
defect: large volumes of ethanol [poorly performed plasmids isolation and
purification], high levels of salinity or unsuspected issue with the gel itself [marker
bands were slightly blurred, even though they were made independently from F and
S samples] (Davis 2012).
Before sending off the genetic material for sequencing, it was required to pick the
best possible sample with potential plasmids that could be sequenced and analysed
for transposase and transposons (Chou & Holmes 2001). As mentioned before,
samples from the second gel were not considered to be a viable option for
sequencing. Samples from the first gel did have a rich genetic material spectre, but
were unlikely to contain plasmids.Therefore, it was decided to sequence samples S1-

38. 38
3 and S2-4, the gel number three [Figure 9, p. 29] – strong single bands most likely
contained plasmids. Plus, sample S2-4 demonstrated an exceptional non-
contaminated DNA throughout the experiment processes. As a result, purified S1-3
and S2-4 were sent off for sequencing.
Final results and conclusion
The sequencing results and analysis revealed credible presence of Sulfolobus genes
in the sea sample which indicated that the “crib” broke the “code” and the
combination of rRNA and tn primers managed to capture partial sequences of
Sulfolobus transposase genes from S2-4 sample. The sequences alignment via the
BLAST revealed multiple matches as well as presence of cognate target genes across
different species of the same genus. These findings can be supported by the fact that
presence of Sulfolobus organisms within English Channel and surrounding waters
was previously observed and recorded by independent studies, which rejects the
possibility of a “lucky coincidence” (Sahan & Muyzer 2008, Baudoux et al. 2012). In
agreement with this finding, we can affirm efficiency of amplification of transposon-
host junctions using rRNA and customized tn primers under proposed conditions.
In respect to the secondary research question, estimates of transposable elements
distribution alter across different aquatic environments: marine, brackish and fresh.
Distribution of transposable elements in a marine environment is univocally larger
than in brackish and/or fresh water environment, based on the fact that the Baffins
Pond sampleturned to be unsuccessfuland the Frog Lakesample did not provide any
convenient material for the search of target genes. The possible explanation could

39. 39
be the environment itself – marine environment is unstable and constantly changing;
English Channel is also a target for constant anthropogenic influence, including
marine transportation links. In contrast, both Baffins Pond and Frog Lake are small-
scale reservoirs with stable conditions and no/low necessity for adaptation to
environmental changes. If this is the case, it could possibly highlight the importance
of transposable elements for evolution and natural selection in aquatic
microorganisms. Nevertheless, it is only aspeculation based on the secondary results
of the study and requires a properly-systematic approach to gather evidence and
prove/disprove the secondary hypothesis.
However, the question “why captured transposase sequences were only partial” is
still open for discussion. The final results gaps indicate the need for extra work and
tests to be conducted to improve the technique. The development of the method
and strategy may be improved in various ways, depending on resources and time
limits. Firstly, gathering more samples across geographically distinct locations could
be a bright idea – it would diversify microbial contents and minimise chances of
hypothetical issues such as contamination, poor quality or human factor [e.g. poorly
performed sampling procedure]. Secondly, primer sets usedfor the experiment could
be modified relying on the flaws of this particular study and/or target specific
organisms directly, e.g. Sulfolobus (Wang & Qian 2009). The PCR strategy and
techniques could also be adjusted to improve the quality of the result DNA – even
though protocols used for this study worked perfectly well, there is always a way to
improve efficiency by manipulating factors and conditions on a reasonable scale
[using previously used techniques as a benchmark]. Lastly, use of several distinct
rRNA primers could be beneficial too or at least worth a try (Frank et al. 2008).

40. 40
However, several obstructions cannot be avoided. For example, some target genomic
regions may not tolerate or ignore insertions of artificially created tn primers
(Barquist et al. 2013). Also, a wide range of transposons and transposase have
specific site preferences which restrain their capabilities of integrating with primers
(Craig 1997). An additional factor that may limit the opportunity of applying this
method are mutated gene regions which may not interact with selected primers
because of unforeseeable differences in genomic patterns (Barquist et al. 2013).
According to successivelaboratory steps and finalresults,it is reasonable to draw the
bottom line and conclude that the proposed method does work the way it was
expected and might be applicable across the majority of biological disciplines after
the eradication of detected limitations. The potential of such method is virtually
limitless and may establish a pioneer way for characterising any aquatic prokaryotic
organism.
Being that the vastmajority of defects may be fixed, it is possibleto apply this method
in the future, along with the decreasing cost of the next generation sequencing tools
(Desai & Jere 2012). The appliance and adaptation of exploitation of tn-host
interactions could be used for a wide range of procedures, including bioremediation,
synthetic biology, astrobiology and bioengineering. Besides, the use of these
methods could be combined with current biotechniques to gather even more fertile
results,leading to a better understanding of complex structures and processes within
microbiological framework. Optimism is not unfounded, as the adaptation of
methods using gene interactions has already become a rapidly growing alternative

41. 41
to classic molecular biology techniques and will likely become increasingly common
in the future (Opijnen et al. 2009).

42. 42
Acknowledgements
First of all, I would like to express my limitless gratitude to Dr James McClellan – this
entire project would not progress without his bright ideas, support and
overwhelmingly positive attitude that motivated me every single day. Also, I would
like to thank my family, friends and girlfriend for a constant sincere support. Special
appreciation to Joey Flight for making days in the lab less stressful and more
productive. The last, but not least, thanks to Dr Niru Nahar who was extremely
supportive throughout the months of challenging but fruitful laboratory work.

43. 43
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