Internship Report

NAME: NABEEL MOHAMMED

IITM 5th Semester
Biological Engineering
BE12B017

Acknowledgement
First I would like to thank Dr. Jackson James, Neural Stem Biology, RGCB for giving me the
opportunity to do an internship within the organization. For me it was a unique experience to
be in RGCB and to study Development of Tetracycline expression system. It also helped me
in understanding mindset of a researcher, importance of doing experiments in a systematic
way and how simple mistakes will result in cascading failures.

I extend my special thanks to Mr. Abdul Rashid, my senior and guide during my internship. I
also would like to thank all the people that worked in the lab of Neural Stem Cell Biology,
RGCB. With their patience and openness they created an enjoyable working environment.
Once again I would like to thank our department especially my guide, Mr. Abdul and
Lab assistants for their guidance and immense support. Furthermore I want to thank all
the scientist and trainee students, with whom I did the labwork.

Table of Contents
1.

INTRODUCTION .................................................................................................................................. 1

2.

ORGANISATION RGCB ................................................................................................................... 2

3.

INTERNSHIP PROJECT ASSIGNED AT RGCB ............................................................................... 3

3.1
OVERALL APPROACH OF TETRACYCLINE SYSTEM DEVELOPMENT ............................................................................ 3
3.2
TASKS INVOLVED IN TETRACYCLINE EXPRESSION SYSTEM ..................................................................................... 6
4. RESEARCH AND BACKGROUND STUDIES .................................................................................... 7
4.1
MODE OF ACTION OF TETRACYCLINE .............................................................................................................. 7
4.2
MECHANISM OF TETRACYCLINE RESISTANCE ..................................................................................................... 8
4.2.1 Enzymatic inactivation of tetracycline. ........................................................................................... 10
4.2.2 Other/unknown mechanisms of resistance. ................................................................................... 10
4.3
REGULATION OF RESISTANCE GENE EXPRESSION ............................................................................................ 11
4.3.1 Efflux genes. .................................................................................................................................... 11
4.3.2 Ribosomal protection. ..................................................................................................................... 11
4.4
INCIDENCE OF TETRACYCLINE RESISTANCE ..................................................................................................... 12
4.4.1 Overview for commensal microorganisms. ..................................................................................... 13
4.4.2 Distribution and Mobility of tet Genes ............................................................................................ 14
4.5
TETRACYCLINE INDUCIBLE EXPRESSION SYSTEM .............................................................................................. 17
4.6
MIRNA .................................................................................................................................................. 18
4.6.1 Biogenesis of miRNA ....................................................................................................................... 19
4.6.2 RNA-induced Silencing Complex ..................................................................................................... 20
4.6.3 Function of miRNA .......................................................................................................................... 21
4.7
RETINA................................................................................................................................................... 22
5. ACTIVITY LOG .................................................................................................................................. 26
6.

EXPERIMENTS PERFORMED AT RGCB ....................................................................................... 29

6.1
PREPARATION OF COMPETENT E. COLI CELLS USING CACL2 ................................................................. 29
6.2
TRANSFORMING COMPETENT CELLS WITH PLASMID DNA .................................................................. 30
6.3
PLASMID ISOLATION USING ALKALINE LYSIS...................................................................................... 33
6.4
AGAROSE GEL ELECTROPHORESIS ............................................................................................ 37
6.5
RESTRICTION DIGESTION ............................................................................................................. 39
6.6
LIGATION.......................................................................................................................................... 40
6.7
SEQUENCING ................................................................................................................................... 40
7. CONCLUSION .................................................................................................................................... 41
8.

REFERENCES .................................................................................................................................... 42

1. INTRODUCTION
When established with diligence and care, tetracycline inducible expression systems can be
invaluable tools in biomedical research. They offer an elegant method to maintain control
over the expression of genes transfected into eukaryotic cells. These systems have evolved
the years to control gene expression in a wide variety of cells including genetically
engineered mice and HeLa cells. Now, the chief mechanism by which E. coli becomes
resistant to high concentrations of tetracycline involves multimeric antiporter proteins known
as Tet proteins which catalyze the outward transport of tetracycline-Mg2+ complexes from the
cytosol. Of these, Tet(A) proteins are the most important in molecular cloning. When Tet(A)
is present in high concentrations, cations are transported from the bacterial cell wall at such a
rate that the membrane becomes depolarised and the viability of the cell is threatened. To
prevent this, expression of Tet(A) is tightly controlled by Tet(R), a helix turn helix repressor
protein. Binding of Tet(R) to the tet-Mg2+ induces a conformational change that reduces the
affinity of the repressor for tet(O) by nine orders of magnitude. The differentials in binding
constants ensure transcription of tet(R) is suppressed in absence of tetracycline and induced
by concentrations of the drug too low to affect protein synthesis.
Various forms of Tet(R) and Tet(O) are used to regulate expression of target genes
transfected into eukaryotic cells. Because all the components repressor, operators and
effectors are prokaryotic in origin, these systems have few if any significant effects on the
host genes. These systems include Tet repression systems, Tet trans-activator system, Tet
reversed activator system and autoregulatory Tet system. The one we will be using is the Tet
trans-activator system.

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2. Organisation RGCB
Rajiv Gandhi Centre for Biotechnology (RGCB) is a premier research institute in India,
exclusively devoted to research in Molecular Biology and Biotechnology. It is located at
Thiruvananthapuram (Trivandrum), the capital city of the state of Kerala in India. This centre
is an autonomous institute under the Department of Biotechnology of the Govt. of India. The
institute has highly focused research departments working on medical biotechnology
(Molecular Medicine, Molecular Reproduction, Molecular Microbiology, Cancer Biology &
Neurobiology) and plant genetic engineering. The Center has a regional facility for Genetic
Fingerprinting, which provides DNA analysis services for forensic & criminal investigations,
paternity disputes, identification of wildlife remains, authentication of plants and seeds
besides a battery of molecular diagnostics for genetic and infectious diseases. RGCB is also a
major provider of laboratory and infrastructure services to other academic and research
institutions.
RGCB has a strength of 25 scientists, 120 Ph.D. students and around 100 research project
staff. The centre has good infrastructural facilities for carrying out cutting-edge research in
the field of Biotechnology. Monetary support of Rs. 100 crores sanctioned by the Govt. of
India in 2008, for a period of 3 years, apart from the yearly allocation of Rs. 25 crores, aims
at making RGCB a world class research centre in the near future.
Neurobiology Stem Cell Biology Lab
The main energies of the lab faculty are devoted towards understanding the signaling
crosstalks involved in maintenance/proliferation of neural stem cells and their fate specific
differentiation. The long term goal is to develop cell replacement therapies to treat refractory
temporal lobe epilepsy and Glaucoma. Recently, the lab faculty has identified a novel Notch
independent Hes-1 activation pathway in a subset of neural progenitors which is critical in its
maintenance and proliferation
In addition to these, they are also involved in characterizing the factors regulating the
excitatory Vs inhibitory fate of differentiating neurons, neural fate specific differentiation of
Umbilical Cord Blood derived mesenchymal stem cells, identification of specific markers for
retinal ganglion cells (RGCs) and development of biodegradable scaffolds for transplantation
studies

3. Internship Project assigned at RGCB

The program assigned to me was development of Tetracycline expression system. Following
section details the tasks associated with above objective.

3.1 Overall approach of tetracycline system development

3.2 Tasks involved in Tetracycline expression system

01. Preparation of competent cells

02. Transformation of cells with vector
03. Inoculation of colony from plate
04. Isolation of plasmid
05. Agarose gel electrophoresis of plasmid (for verification)
06. Restriction digestion of vector
07. Agarose gel electrophoresis (for elution)
08. Cutting appropriate piece and gel elution
09. Ligation of eluted vector and insert
10. Transformation of new plasmid
11. Inoculation of new plasmid
12. Plasmid Isolation
13. Preparation of stock solution of plasmid
14. Agarose gel electrophoresis (for verification)
15. Restriction digestion of plasmid
16. Agarose gel electrophoresis (for verification)
17. Sequencing of plasmid

4. Research and Background Studies

Tetracyclines are a group of broad-spectrum antibiotics. They are so named for their four
(tetra-) hydrocarbon rings (-cycl-) derivation (-ine). To be specific, they are defined as
"a subclass of polyketides having an octahydrotetracene-2-carboxamide skeleton".
The tetracyclines, discovered in the 1940s, are a family of antibiotics. They inhibit protein
synthesis by thwarting the attachment of aminoacyl-tRNA to the ribosomal acceptor (A) site.
Tetracyclines are broad-spectrum agents which imply that they act against a wide range of
gram-positive and gram-negative bacteria, atypical organisms such as chlamydiae,
mycoplasmas, and rickettsiae, and protozoan parasites. These beneficial anti-microbial
properties and the absence of major adverse side-effects have led to their extensive use in the
treatment of human and animal infections. It is a reputable fact that tetracyclines inhibit
bacterial protein synthesis by preventing the association of aminoacyl-tRNA with bacterial
ribosomes. Therefore, to work against their targets these molecules need to traverse one or
more membrane systems depending on whether the susceptible organism is gram positive or
gram negative. Hence, a discussion of the mode of action of tetracyclines requires
consideration of uptake and ribosomal binding mechanisms.

4.1 Mode of action of Tetracycline

Tetracyclines pass through the outer membrane of gram-negative enteric bacteria through the
OmpF and OmpC porin channels, as cation-tetracycline coordination complexes(Chopra,
Hawkey et al. 1992). The cationic metal ion-antibiotic complex is attracted by the Donnan
potential(potential caused by ions separated due to a semi-permeable barrier) across the outer
membrane leading to accumulation in the periplasm. Here, the metal ion-tetracycline
complex dissociates to give uncharged tetracycline which as a weakly lipophilic molecule is
able to diffuse through the lipid bilayer regions of the inner membrane. Likewise, the
electrically neutral, lipophilic form is assumed to be the species transferred across the
cytoplasmic membrane of gram-positive bacteria. Uptake of tetracyclines across the
cytoplasmic membrane is energy dependent and driven by the pH difference component of
the proton motive force. Within the cytoplasm, tetracycline molecules are likely to become
chelated due to the relatively higher internal pH and divalent metal ion concentrations
compared to those outside the cell(Schnappinger and Hillen 1996). Association of
tetracyclines with the ribosome is reversible which explains why the effects of these
antibiotics are more likely to prevent bacterial reproduction than to kill them.
Several studies have denoted a single, high-affinity binding site for tetracyclines in the
ribosomal 30S subunit. Elucidation of the studies referred to above is made difficult by the
observation that binding of tetracycline to the ribosome seems to cause wide-ranging
structural changes in 16 S rRNA, a component of the 30S subunit(Noah, Dolan et al. 1999).
Furthermore, the light based methods used in these studies are subject to the limitation that
7

tetracycline photoproducts capable of reacting further with the ribosomes may be generated
upon irradiation(Oehler, Polacek et al. 1997).
The absence of major anti-eukaryotic activity helps clarify the selective antimicrobial action
of the tetracyclines. At the molecular level, this is due to the relatively weak inhibition of
protein synthesis championed by 80S ribosomes as well as the poor accumulation of these
antibiotics by mammalian cells. However, it is a well known fact that tetracyclines inhibit
protein syntheses in mitochondria, (Riesbeck, Bredberg et al. 1990) possibly due to the presence
of 70S ribosomes in these organelles. Although it has been recognized for some time that the
spectrum of activity of tetracyclines encompasses various protozoan parasites such as P.
falciparum, such antiparasitic activity can be explained in several cases by the discovery that
certain organisms, e.g., P. falciparum, contain mitochondria. However, a number of other
protozoa lacking mitochondria have, all the same, found to be susceptible to tetracyclines
(Edlind 1991). Nonetheless, the relevant detail to take from these findings is that tetracycline
does not act on eukaryotic cells to an extent and thus any effects it has on a transfected
eukaryotic cell can be traced back to the prokayotic DNA transfected into it.

4.2 Mechanism of tetracycline resistance

Prior to the mid-1950s, the majority of pathogenic bacteria were susceptible to
tetracyclines(Levy and Marshall 2004). Studies of endemic environmental bacteria, symbolic
of populations existing before the widespread use of tetracyclines also support the outlook
that the emergence of tetracycline resistance is a relatively modern event that has followed
the clinical, veterinary, and agricultural use of these agents. Indeed, resistance to tetracyclines
have emerged in pathogenic bacteria chiefly due to genetic acquisition of tet genes.
In 1980, Mendez et al. examined the genetic heterogeneity of tetracycline resistance
determinants induced by plasmids from members of the Enterobacteriaceae and
Pseudomonadaceae (Mendez, Tachibana et al. 1980). The usage of restriction enzyme
analysis, DNA-DNA hybridization, and expression of resistance to tetracycline and various
analogs was done to categorize the tetracycline-resistant (Tcr) plasmids. At present, in the
viewpoint of Stuart Levy, two genes are considered related (i.e., of the same class) and given
the same gene designation if they have 80% of their amino acid sequences in common.
Hence, it follows that two genes are considered different from each other if they have 79%
amino acid sequence identity(Levy, McMurry et al. 1999). For most genes, only one
representative of each class have been sequenced making comparisons easier to perform. An
exception to this rule is the tet(M) gene, which has been sequenced from a number of
species(Roberts 1996). The number of tet genes have long since reached the end of our
alphabet and nowadays, numbers are being assigned to accommodate new tet genes(Levy,
McMurry et al. 1999). Moreover, chiefly due to Levys efforts, it has been agreed to
coordinate the assignment of proposed numbers for a new tetracycline resistance gene so as
to prevent two distinct tet genes from being assigned the same numbers or two related genes
(80% identity) being assigned different numbers(Levy, McMurry et al. 1999).
As a result, twenty-nine different tetracycline resistance (tet) genes and three oxytetracycline
resistance (otr) genes have been characterized. There is no inherent difference between a
tetracycline and an oxytetracycline resistance gene. The oxytetracycline genes were first
identified in oxytetracycline-producing organisms, and thus the nomenclature reflects the
organisms first shown to carry the particular gene. Eighteen of the tet genes and one of
8

the otr genes code for efflux pumps, and seven of the tet genes and one of
the otr genes otr(A) code for ribosomal protection proteins. An uncharacterized gene has also
been described in a gram-negative species, and four different ribosomal protection genes,
from streptococci, have been cloned using degenerate PCR primers. Whether these genes are
related to some of the newer tet genes is not clear although the fact that the new ribosomal
protection genes did not hybridize with tet(M), tet(O), tetP(B), tet(Q), tet(S), or tet(T),
suggest that at least some of the four could be novel. It is interesting to note that the tet(X)
gene encodes an enzyme which modifies and inactivates the tetracycline molecule. However,
as of now, it does not have much medical relevance as it both requires oxygen to function and
is found only in strict anaerobes.
The efflux proteins are the best studied of the Tet proteins. The genes encoding then belong
to the major facilitator superfamily (MFS), whose products include over 300 individual
proteins. All the tet efflux genes code for membrane-associated proteins which, as the name
efflux suggests, are involved in the export of tetracycline from the cell. Export of tetracycline
results in the reduction of intracellular drug concentration and thereby protects the ribosomes
within the cell. Efflux genes are found in both gram-positive and gram-negative species.
Most of these efflux proteins appear to reside in the lipid bilayer, with the hydrophilic aminoacid loops protruding into the periplasmic and cytoplasmic space. The efflux proteins work
by exchanging a proton for a tetracycline-cation complex against a concentration gradient.
Tetracycline efflux proteins have amino acid and protein structure similarities with other
efflux proteins involved in multiple-drug resistance, quaternary ammonium resistance, and
chloramphenicol and quinolone resistance, including methylenomycin A (MetA)
from Streptomyces coelicolor, aminotriazole transport (Atr1) fromSaccharomyces, and
arabinose transport (AraB) from Escherichia coli.
The gram-negative efflux genes are widely disseminated and are typically associated with
large plasmids. Most of these plasmids are conjugative and come from a number of different
plasmid incompatibility groups. These plasmids often carry other antibiotic resistance genes,
heavy metal resistance genes, and/or pathogenic factors such as toxins(Falkow 1975). This is
extremely important as it means that selection for any of these factors selects for the plasmid.
This phenomenon of cross-selection has contributed to the dramatic increase in the number
multiple-drug-resistant bacteria over the last 40 years.
Ribosomal protection proteins are cytoplasmic proteins that, as their name implies, protect
the ribosomes from the action of tetracycline. They also confer resistance to doxycycline and
minocycline. A notable fact about them is that they bestow a wider spectrum of resistance to
tetracyclines than is seen with bacteria that carry tetracycline efflux proteins, with the
exception of Tet(B). The Tet(M), Tet(O), and OtrA proteins reduce the susceptibility of
ribosomes to the action of tetracyclines. The mechanism of ribosomal protection works in
vivo and in vitro, unlike the action of efflux proteins; which require intact membranes to
function.
The Tet(M) and Tet(O) proteins are the most comprehensively characterized of the ribosomal
protection group. They have been shown to have ribosome-dependent GTPase activity.
However, the Tet(M) protein could not replace the function of the EF-G(Elongation Factor
Growth) protein in an E. coli isolate with a temperature-sensitive EF-G protein, at the
nonpermissive temperature. The Tet(M) protein does not replace either the EF-G or EF-Tu
proteins in an in vitro protein synthesis assay(Burdett 1996). The Tet(O) protein binds GDP
and GTP. Site-directed mutations in the Tet(O) protein, which reduce the binding of GTP,
were correlated with reduction in the susceptibility to tetracycline in isolates. This suggests
9

that the GTP binding is important to the function of the Tet(O) protein(Taylor, Trieber et al.
1998).
It was found that the Tet(M) protein allows the aminoacyl-tRNA to bind to the acceptor site
of the ribosome in the presence of tetracycline concentrations that normally inhibit
translation(Burdett 1996). In the presence of the Tet(M) protein, tetracycline is apparently
released from the ribosomes. In the presence of either the Tet(M) or the Tet(O) protein,
tetracycline binding to the ribosomes is reduced when GTP but not GDP is present. Energy
from GTP hydrolysis released the tetracycline from the ribosome when a nonhydrolyzabe
GTP analog was used(Burdett 1996). Although only two of the proteins from this group have
been extensively examined, it has been assumed that the other proteins in the ribosomal
protection group [Tet(S), Tet(T), Tet(Q), TetB(P), Tet(W), and Otr(A)] have GTPase
activity and interact with tetracycline and the ribosomes in similar ways to those described
for the Tet(M) and Tet(O) proteins, because of the similarities at the amino acid sequence
level.
The current data suggests that the ribosomal protection proteins bind to the ribosome. This
instigates an alteration in ribosomal conformation which prevents tetracycline from binding
to the ribosome, without altering or terminating protein synthesis. The hydrolysis of GTP is
hypothesised to possibly provide the energy for the ribosomal conformational change. The
ribosomal protection proteins also need to dissociate from the ribosome to allow EF-G to
bind, since they have overlapping binding sites on the ribosome.

4.2.1 Enzymatic inactivation of tetracycline.

The tet(X) gene encodes the only example of tetracycline resistance due to enzymatic
alteration of tetracycline(Speer, Bedzyk et al. 1991). Two closely related
anaerobic Bacteroides transposons containing the tet(X) gene have been described(Speer,
Bedzyk et al. 1991). The tet(X) gene was found because it is linked to erm(F), which codes
for a rRNA methylase gene. The erm(F) gene was cloned into E. coli, and the clones were
found to confer tetracycline resistance in E. coli when grown aerobically. The tet(X) gene
product is a cytoplasmic protein that chemically modifies tetracycline in the presence of both
oxygen and NADPH. Sequence analysis has indicated that this protein has amino acid
homology with other NADPH-requiring oxido-reductases and should not be able to function
in the natural anaerobic Bacteroides host(Speer, Bedzyk et al. 1991). It has not been found
outside Bacteroides. To date, no surveys have been conducted to assess the distribution of
the tet(X) gene. Thus, even though the transposon carrying tet(X) and linked erm(F) is
thought to be of gram-positive aerobic or facultative origin, an acknowledged ancestor has
yet to be identified.

4.2.2 Other/unknown mechanisms of resistance.

The tet(U) gene confers low-level tetracycline resistance(Ridenhour, Fletcher et al. 1996).
This gene encodes a which is smaller than both the efflux proteins and the ribosomal proteins.
There is 21% similarity over the 105 amino acids between the Tet(U) and Tet(M) proteins.
These similarities do not include the sequences thought to play a role in resistance in the
Tet(M) and related proteins. Thus, the sequence is not really similar to either the efflux or
ribosomal protection genes, and the mechanism is unknown.
Similarly, the mechanism of resistance of the otr(C) gene from Streptomyces has not been
determined because it has not yet been sequenced. It has been cogitated that the otr(C) gene
10

does not code for either an efflux or ribosomal protection protein. Whether otr(C) encodes an
inactivation enzyme, similar to tet(X), or whether it has a novel mechanism of resistance
like tet(U) has not yet been determined.

4.3 Regulation of Resistance Gene Expression

4.3.1 Efflux genes.
The gram-negative efflux determinants consist of two genes, one coding for an efflux protein
and one coding for a repressor protein both of which are regulated by tetracycline. The two
genes are oriented divergently and share a central regulatory region with overlapping
promoters and operators (Hillen and Berens 1994). In the absence of tetracycline, the
repressor protein occurs as a homodimer, which binds two -helixturn-helix motifs to the
two tandemly orientated tet operators (Hillen and Berens 1994). This blocks transcription of
the structural genes for both the repressor and the efflux protein. Initiation of the system
occurs when a tetracycline-Mg2+ complex enters the cell and binds to the repressor protein.
Tetracycline binding changes the conformation of the repressor so that it can no longer bind
to the DNA operator region. Only nanomolar concentrations of tetracycline are needed for
binding to the repressor protein. This system is the most sensitive effector-inducible
transcription regulation system which has been discovered so far. After the repressor binds
the tetracycline-Mg2+ complex, transcription of the efflux and repressor genes occurs. This is
a relatively rapid process. The tet gene in Tn10 (transposable element) is differentially
regulated so that the repressor protein is synthesized before the efflux protein is expressed
(Hillen and Berens 1994). The repressor protein will rebind to the DNA only when there is
insufficient tetracycline (smaller than nanomolar amounts) present in the cell. This type of
regulation in all probability occurs with all the gram-negative efflux genes. Crystallography
has shown the DNA-binding domain in the repressor molecule and that conformational
changes in the repressor protein occur in the presence of tetracycline complexed with Mg2+.
The tetracycline-binding pocket and the interaction between tetracycline and the repressor
protein have also been characterized (Hillen and Berens 1994).
Three different strains of Haemophilus parainfluenzae were shown to carry
constitutively(constant and active) expressed Tn10 (Heuer, Hickman et al. 1987).
Subsequently, it was shown that a truncated non-functional repressor protein due to a
frameshift mutation in the repressor gene was present(Heuer, Hickman et al. 1987) which
resulted in the constitutive expression of the Tet(B) protein. However, when a functional
repressor was added to the cell, the tet(B) gene was inducible and regulated normally. The
incidence of defective repressors in nature outside the genus Haemophilus has not been
examined.

4.3.2 Ribosomal protection.

The expression of both Tet(M) and Tet(O) proteins appears to be regulated. It has been
suggested (Wang and Taylor 1991) that the 400-bp region directly upstream from the coding
region of the tet(O) gene was needed for full expression of the gene; however, the function of
the region is not understood(Taylor and Chau 1996). It was also reported that the amount of
Tet(M) protein increased when streptococci carrying the determinant were exposed to
tetracycline(Burdett 1991). Similarly, it was found that preexposure to subinhibitory
concentrations of tetracycline in S. aureus strains carrying the tet(M) gene resulted in both an
increase in tetracycline resistance and an increase in the level of mRNA transcripts for tet(M)
11

(Nesin, Svec et al. 1990). Scientists have reported a stem-loop structure in the upstream
region of the structural gene from Tn916, and both short and long transcripts were found by
Northern blot analyses, similar to descriptions for attenuation of mRNA transcription of
gram-positive proteins(Su, He et al. 1992). Based on the DNA sequences from the upstream
region in the Ureaplasma urealyticum tet(M) sequence, a similar stem-loop structure to that
described for Tn916 would be possible.

4.4 Incidence of Tetracycline Resistance

Most work on bacterial resistance have either been conducted on pathogenic bacteria or
opportunistic bacteria. Opportunistic bacteria are part of the host's normal flora and can cause
disease when they leave their normal sites or when the microenvironment in the host is
drastically altered. The majority of tet genes in bacteria have been associated with mobile
plasmids, transposons, conjugative transposons and integrons (gene cassettes). This has
enabled the tet genes to move from species to species and into a wide range of genera by
conjugation. It has been speculated that some genes, such as tet(E), may have a more limited
host range as they are located on non-mobile plasmids, which drastically diminishes
opportunities for transfer to other species and genera(DePaola and Roberts 1995).
In 1953, the first tetracycline-resistant bacterium, Shigella dysenteriae, was isolated(Falkow
1975). The first multiple-drug resistant Shigella was isolated in 1955. This later isolate was
resistant to tetracycline, streptomycin, and chloramphenicol and represented 0.02% of the
isolates tested(Falkow 1975). By 1960, multiple-drug-resistant Shigella represented roughly
10% of the strains tested in Japan.(Falkow 1975). The increase in multiple-drug resistant
strains has continued to the present day. Studies showed that over 60% of the S.
flexneri strains isolated between 1988 and 1993 were resistant to tetracycline, streptomycin,
and chloramphenicol, which is the same combination of antibiotic resistance determinants
found in the 1953 S. dysenteriae isolate. It was demonstrated that these antibiotic-resistant
bacteria could transfer all their antibiotic-resistant phenotypes to susceptible isolates by cocultivation(Watanabe 1963). This transfer was dependent on direct contact of the viable
growing bacteria. We now know that the Japanese studies were the first reports of
tetracycline resistance genes carried on conjugative R-plasmids. These tetracycline resistance
genes conferred efflux of tetracycline from the cell and encoded the first of the three different
types of tetracycline resistance mechanisms to be found in bacteria.
Isolation of Salmonella enterica serovar Typhimurium (Typhoid causing bacteria) has
become more widespread in recent years in both human and animal sources, and the isolates
have been genetically characterized. Many of the isolates are multiple-drug resistant and
carry a class 1 integron containing an assortment of different antibiotic resistance genes
including those encoding resistance to tetracycline. In a recent study, 10 human and 8
nonhuman isolates carried the tet(G) gene, along with genes conferring resistance to one or
more other antibiotics including ampicillin, chloramphenicol, streptomycin, spectinomycin,
and sulfonamide. One human isolate carried the tet(A) gene, and three carried tet(B) in place
of the tet(G) gene. Multiple-drug resistance, which includes Tcr, has been identified in an
increasing number of gram-negative pathogens and opportunistic bacteria.
Gram-positive species have also acquired Tcr, especially those that are multiple-drug
resistant. A 1994 study found that approximately 90% of the methicillinresistant Staphylococcus aureus, 70% of Streptococcus agalactiae, 70% of multiple-drug
resistant Enterococcus faecalis, and 60% of the multiple-drug resistantStreptococcus
pneumoniae strains were now Tcr(Goldstein, Kitzis et al. 1994).
12

Gram-negative tet genes are those which have been found only in gram-negative bacteria.
These genes have higher G+C contents (>40%) than those of gram-positive origin. All of the
gram-negative tet genes encode efflux proteins which work by expelling tetracycline from the
cell. Hence, they do not express well if moved into gram-positive hosts which have a vastly
different membrane structure. Most of the gram-negative tet genes are regulated by a
repressor, which is transcribed in the opposite direction from the structural gene. Grampositive tet genes are those which are usually found in gram-positive species but, more
importantly, have relatively low G+C contents (<35%). Contrary to the gram-negative tet
genes, the gram-positive tet genes are found in an increasing number of gram-negative
species, including anaerobes. This is especially true with the tet(M) gene, which has been
identified in clinical isolates from several gram-negative genera and gram-positive genera.
Furthermore, the tet(M) gene has been conjugally transferred in the laboratory to an even
larger group of species and genera than has been found in natural isolates.
It was found that long-term use of tetracycline selects not only for tetracycline-resistant
gram-negative bacteria but also for multiple-drug-resistant gram-negative species(Levy
2002). Tcr genes in both gram-positive and gram-negative species are often found on the
same units (plasmids, transposons, or integrons) as other antibiotic resistance genes. For
example, all chloramphenicol-resistant (Cmr) Haemophilus influenzae strains isolated in the
1970s and 1980s were also Tcr. One hypothesis was that the first H. influenzae strains to
obtain Cmr had acquired the Tcr Cmr transposon, which was then passed between strains and
species. In contrast, some H. influenzae strains obtained tetracycline resistance transposons
without the chloramphenicol gene and thus passed on only the Tcr..
Scientists have hypothesized that the transposon carrying the tet(M) gene, as typified by
Tn916, was the original gram-positive conjugative transposon(Flannagan, Zitzow et al.
1994). It is suggested that over time other antibiotic resistance genes were inserted directly
into this family of transposons, creating larger units carrying two to four different antibiotic
resistance genes. In addition, multiple conjugative transposons, which have a single complete
transposon inserted within another transposon, have been described in some of the cocci.
These can either transfer as a single unit or the inserted transposon can be transferred
independantly, giving flexibility for transfer of antibiotic resistance genes. Selection for any
antibiotic on these multiple-drug-resistant units normally selects for the entire unit and also
helps explain the isolation of Tcr bacteria in children even though tetracycline is not used in
this age group.
Obligatory intracellular pathogens such as Chlamydia and Rickettsia have not yet acquired
tetracycline resistance. Since these bacteria grow only inside cells, it would require that cells
be infected with two genera to allow gene exchange into the obligate intercellular pathogen.
Mutations to increased tetracycline resistance would be more likely to occur in such
intracellular bacteria. A few reports have described heterotypic tetracycline resistance inC.
trachomatis when grown at high density, but there was no clear correlation of the phenotype
to isolates from patients who did not respond to tetracycline therapy. On further examination,
the phenotype was not stably transferred and was most probably an artifact of the growth
conditions (Jones et al., 30th ICAAC).

4.4.1 Overview for commensal microorganisms.

The commensal flora consist of microorganisms which are present in and on surfaces of a
host and are not thought to cause disease. These organisms are often beneficial to the host,
supplying nutrients and inhibiting the growth of potential pathogens by preventing them from
13

becoming established. Thus, it is not suprising that these bacteria have the same tet genes,
plasmids, transposons, conjugative transposons, and integrons as their disease-producing
counterparts among the opportunistic and pathogenic bacteria. Many oral viridans
streptococci have acquired tet(M), tet(O), tet(L) or tet(K), as have the pathogenic
streptococcal species S. pneumonia, etc. Most people today carry Tcr viridans streptococci in
their mouth regardless of use of tetracycline therapy or age, while Tcr S. pneumoniae and S.
pyogenes are significantly less common in most populations.
An additional observation is that, as time progresses, the gram-positive commensal bacteria
have evolved from carrying single tet genes to carrying multiple tet genes. The
differing tet genes can follow either the same mechanism of action (efflux or ribosomal
protection), or different mechanisms of action (efflux and ribosomal protection), similar to
what pathogenic and opportunistic species do. The carriage of multiple tet genes of different
classes is commonly found in individual gram-positive isolates and in Mycobacterium spp.
and Streptomyces spp. but is uncommon in facultative gram-negative bacteria, especially
enteric species. The reason for this is unknown, but a similar situation exists for the carriage
of other antibiotic resistance genes.
To stimulate the study of commensal bacteria, a new group recently came together to focus
on antibiotic-resistant commensal bacteria and was named the Reservoirs of Antibiotic
Resistance or (ROAR) project. The purpose of the group is to promote the study of carriage
of antibiotic-resistant bacteria in humans, during food production and agricultural processes,
and in the environment. The ROAR project is providing a source of information on resistance
in commensal bacteria and can be found at. http://www.roar.antibiotic.org. Another Web site,
which can be accessed from the ROAR site or directly, is The Alliance for the Prudent Use of
Antibiotics (APUA) (http://www.APUA.org). This is a nonprofit organization whose purpose
is to promote proper antibiotic use and curb antibiotic resistance worldwide.
In general, it has been found that most disease-producing species of gram-negative genera
carry the same tetracycline resistance genes as do the commensal species within the same
genus. In view of the above observations, it has been proposed that commensal bacteria act as
a reservoir for tet and other antibiotic resistance genes found in, human pathogens and are
thus very important in our understanding of how antibiotic resistance genes are maintained
and spread through bacterial populations.

4.4.2 Distribution and Mobility of tet Genes

4.4.2.1 Overview.
The tet genes have been unearthed in a plethora of bacteria isolated from humans, animals,
and the environment. The preponderance of the tet genes are associated with either
conjugative or mobilizable elements, which may, to some extent, explain their wide
distribution among bacterial species(Jones, Osborne et al. 1992). The gram-negative tet efflux
genes are found on transposons inserted into an assorted group of plasmids from an array of
incompatibility groups(Jones, Osborne et al. 1992). Gram-positive efflux genes are associated
with small plasmids. The ribosomal protection genes tet(S) and tet(O) can be found on
conjugative plasmids, or in the chromosome, where they are not self-mobile(Charpentier,
Gerbaud et al. 1993). The tet(M) and tet(Q) genes are generally associated with conjugative
chromosomal elements, which code for their own transfer(Li, Shoemaker et al. 1995). These
conjugative transposons transfer mobilizable plasmids to other isolates and species and even
unlinked genomic DNA(Li, Shoemaker et al. 1995). Genes in the tet(Q) operon have been
identified which mediate excision and circularization of discrete nonadjacent segments of
14

chromosomal DNA in Bacteroides. The transfer origin (oriT) region of one of the
Bacteroides conjugative transposons has been located near its middle(Li, Shoemaker et al.
1995). DNA transfer is tetracycline regulated and mediated by at least three regulatory genes
including a putative sensor (rteA), a putative regulator (rteB), and a third gene, rteC, which
seems to stimulate transfer in an unknown fashion(Li, Shoemaker et al. 1995). An alternate
mechanism proposed for tetracycline regulation of conjugation of the Tn916 family of
elements has suggested that tetracycline increases the number of circular intermediates
present in the cell leading to more transconjugants. Thus, a gene can be induced to increase
conjugal transfer by the presence of low doses of tetracycline.
Studies have found a correlation between the plasmid incompatibility group and the
particular tet genes carried by the plasmid. They suggest that some of the tet genes may have
become genetically linked to specific incompatibility and/or replication genes and thus the
distribution of these genes could reflect the occurrence of particular incompatibility groups in
particular genera or species. This hypothesis has not been thoroughly examined and requires
further examination.

4.4.2.2 Gram-negative bacteria.

Currently 39 genera of gram-negative bacteria and 23 genera of gram-positive bacteria and
related genera have been described in which the mechanism of tetracycline resistance has
been determined. Other uncharacterized tet genes probably exist since studies have identified
tetracycline-resistant isolates which do not carry any of the known tet genes(Jones, Osborne
et al. 1992; DePaola and Roberts 1995). New tet genes are continuing to be described, and
new genera have been identified which carry acquired tet genes.
The tet(A), tet(B), tet(C), tet(D), tet(E), tet(G), tet(H), tet(I), tet(J), tet(Y), tet(30), and tet(31)
genes are found exclusively in gram-negative. Most of these genera belong to the facultative
enteric group. The tet(B) gene has the widest host range among gram-negative species and
has been identified in various species. The tet(B) gene is found on conjugative plasmids
in Actinobacillus and Haemophilus. Interestingly, it has been shown that it is possible to
transfer the tet(B)-carrying plasmid from A. actinomycetemcomitans to H. influenzae (Roe,
Braham et al. 1995). The tet(B) gene is not mobile in the small number of Moraxella
and Treponema isolates examined, but it would nonetheless be fascinating to determine
whether either A. actinomycetemcomitans or Haemophilus spp. could transfer their tet(B)
genes into either of these genera and if the transconjugants could then transfer the tet(B)
gene(Roe, Braham et al. 1995).
Of the species which carry the tet(M) gene, some have complete conjugative elements which
are mobile, while another species has a complete conjugative element integrated into a
conjugative plasmid. Other species carry nonconjugative incomplete elements in their
chromosome, like Neisseria spp. or incomplete elements on conjugative plasmids, like N.
gonorrhoeae(Roberts 1989) . In N. gonorrhoeae, the incomplete transposons are associated
with 25.2-MDa conjugative plasmids. The 25.2-MDa conjugative plasmids have one of two
different deletions of the tet(M) transposon(Roberts 1989). One plasmid has a deletion
downstream of a HincII site while the second plasmid type has a deletion of over 800 bp
upstream of the HincII site(Roberts 1989). Both types of plasmid confer resistance to 16 g
of tetracycline/ml and can transfer to other N. gonorrhoeae strains at frequencies ranging
from 101 to 109 per recipient(Roberts 1989). Even though N. meningitidis has been found
naturally with only one of the two plasmid types, both are readily transferred by conjugation
into N. meningitidis. The 25.2-MDa plasmids can be stably transferred into a number of the
commensal Neisseria spp. in the laboratory but, to date, only two species have been found
15

that naturally carry the tet(M) gene. In all these isolates, an incomplete Tet(M) transposon
located in the chromosome rather than on the 25.2-MDa plasmid has been found. This
illustrates that plasmids or genes can be moved in the laboratory, resulting in stable
transconjugants, but similar strains are not found in natural isolates.
Both 25.2-MDa plasmids can be transferred in the laboratory into Haemophilus spp. (Knapp,
Johnson et al. 1988). Once the Neisseria 25.2-MDa plasmid has been transferred
into Haemophilus spp., it is easily transferred among species within the
genus Haemophilus but not back to Neisseria spp. (Roberts and Knapp 1988). Similarly,
an H. ducreyi plasmid which carries the complete Tet(M) transposon can be transferred
among Haemophilus spp. but not outside the genus(Roberts 1989). This type of plasmid
incompatibility may influence the spread of particular tet genes into new genera.
The tet(Q) gene was first described in human colonic Bacteroides spp. and is normally
associated with conjugative elements. The tet(Q) gene has been found in seven gram-positive
genera and six gram-negative genera; it is possible that the host range of the tet(Q) gene
might be similar to that found for the tet(M) gene.
The number of different tet genes found in a particular gram-negative genus varies from
one tet gene carrying either tet(B) (efflux), tet(M), or tet(Q) (both ribosomal protection), to a
maximum of seven different tet efflux genes found in the genus Escherichia. However, it was
discovered that no E. coli plasmids carried more than one type of tet gene(Jones, Osborne et
al. 1992) (Mendez, Tachibana et al. 1980). In contrast, Streptomyces and gram-positive
genera often have individual isolates that carry multiple genes coding for the same
antimicrobial resistance including tetracycline. It is clear that many environmental, food and
animal isolates are tetracycline resistant. However, these bacteria have not yet been as
extensively examined as those associated with human disease. Little work has been done to
elucidate why some genera carry only a single tet gene, while members of other genera can
carry a variety of different tet genes. More work is required to better understand the factors,
which influence not only whether a particular genus or species of bacteria will carry
tetracycline resistance genes but also whether they will encode efflux, ribosomal protection,
or both.
The incompatibility groups of gram-negative plasmids, which determines their bacterial host
range, has been studied. Plasmid host ranges vary from very restrictive, such as with the large
conjugative Haemophilus R-plasmids, which do not readily survive outside their own genus,
to broad, which allows the plasmid to survive in diverse host backgrounds. These differences
in plasmid host range may influence the spread of particular tet genes associated with them.
Gram-negative anaerobic genera and some nonenteric gram-negative genera most commonly
or exclusively carry ribosomal protection genes. Eight gram-negative genera currently carry
tet(M) genes, seven carry tet(Q), five carry tet(W) genes, two carrytet(O), two carry tet(K),
and one each carries tet(H), tet(I), tet(J), tet (Y), tet(30), and tet(31). Based on their low G+C
content and their regulation, tet(K),tet(L), tet(M), tet(O), tet(P), tet(S), tet(T), tet(Q), tet(W),
and maybe tet(Z) are thought to be gram-positive origin. This strengthens the hypothesis that
antibiotic resistance genes from gram-positive species, especially the ribosomal protection
genes, have been exchanged throughout the bacterial population without regard to species or
genus and can be successfully integrated and expressed in a variety of bacterial host
backgrounds.

16

4.4.2.3 Gram-positive bacteria.

Seventeen Gram-positive genera, two cell wall-free genera, Actinomyces, Nocadia,
Mycobacterium, and Streptomyces are all known to carry acknowledged tetracycline
resistance genes. However, just as it was with gram-negative isolates, not all tetracyclineresistant gram-positive bacteria have been correlated with possession of specific
known tet genes. A total of 18 genera carry the tet(M) gene, 11 carry tet(K), 10 carry tet(L), 7
carry tet(Q), 5 carry tet(O), 2 carry tet(S), and 1 carries tet(P). Recently, Bifidobacterium spp.
have been shown to carry the tet(W) gene (Scott, Melville et al. 2000)and
Corynebacterium carries either tet(M) or the newly described tet(Z) (Levy, McMurry et al.
1999). The new tet(U) gene is found in Enterococcus, the tet(T) gene is found
in Streptococcus, and the tet(V) gene is found in Mycobacterium smegmatis.
The tet(K) and tet(L) genes have been found in rapidly growing Mycobacterium, Norcardia,
and Streptomyces spp. isolated from patients(Pang, Brown et al. 1994). This is the first time
that acquisition of an antibiotic-resistance determinant has been documented in
Mycobacterium and Nocardia spp., and it suggests that gene exchange between tetracyclineresistant gram-positive bacteria, Mycobacterium spp., Nocardia spp., and Streptomyces spp.
has occurred. The hypothesis that Streptomyces spp. exchange antibiotic resistance genes
with other genera is strengthened by the finding of the otr genes, from industrial
Streptomyces spp., in clinical isolates of Mycobacterium and Streptomyces spp(Pang, Brown
et al. 1994). This is consistent with the hypothesis that some of the tet genes could be
ancestrally related to genes found in the antibiotic-producing Streptomyces spp(Benveniste
and Davies 1973).
The tet(M) gene is often associated with a conjugative element of the Tn916Tn1545 family(Clewell, Flannagan et al. 1995). This group of elements form non-replicating
circular intermediates, which are essential for both intracellular transposition and intercellular
conjugative transfer. Both types of movements occur by an excision-integration process,
where excision and formation of a covalently closed circular molecule precedes movement of
the conjugative element. In both Tn916 and Tn1545, imperfect inverted repeats (20 of 26 bp)
are present at the ends of the transposon and integration occurs without duplication of the
target DNA sequence. More recently, scientists have shown that the number of circular
intermediates varies in different Enterococcus faecalis strains. Their work indicates that the
number of circular intermediates influences the frequency of conjugation while others have
demonstrated that the number of circular intermediates increased when the strains were
grown in the presence of tetracycline.

4.5 Tetracycline Inducible Expression System

The study of gene function in complex genetic environments such as mammalian cells would
greatly profit from systems that would allow stringent control of the expression of individual
genes(Gossen and Bujard 1992). Ideally, such systems would not only mediate an "on/off"
situation of gene activity but also would permit limited expression at a defined level.
Attempts to control gene activity by various inducible eukaryotic promoters responsive to, for
example, heavy metal ions, heat shock, or hormones have generally suffered from leakiness
of the inactive state (e.g.the metallothionein promoter) or from pleiotropic effects caused by
17

the inducing principles themselves, such as elevated temperature or glucocorticoid hormone

action.In search of regulatory systems that do not rely on endogenous control elements,
several groups, including ours, have demonstrated that the lac repressor/operator/inducer
system of Escherichia coli functions in mammalian cells. Three basically different
approaches have been described: (i) prevention of transcription initiation by properly placed
lac operators at promoter sites, (ii) blockage of transcribing RNA polymerase II during
elongation by a lac repressor/operator complex, and (iii) activation of a promoter responsive
to a fusion between lacR and the activating domain of virion protein 16 (VP16) of herpes
simplex virus (HSV).At present, however, the utility of the lacR/O-based systems in
mammalian cells is limited since the inducer isopropyl /B-D-thiogalactopyranoside (IPTG),
despite its rapid uptake and intracellular stability, acts rather slowly and inefficiently,
resulting in only moderate induction(Gossen and Bujard 1992). Nevertheless, an interesting
conditional mutant of a lacR-VP16 fusion has been described. It activates a minimal promoter
-'1000-fold at elevated temperatures in the presence of IPTG. The temperature dependence
and the inherent IPTGrelated problems, however, may once again limit this approach. Here
we describe a control system that in HeLa cells allows regulation of expression of an
individual gene over up to five orders of magnitude(Gossen and Bujard 1992). This system is
based on regulatory elements of the TnlO-specified tetracycline-resistance operon of E. coli,
in which transcription of resistancemediating genes is negatively regulated by the tetracycline
repressor (tetR). In the presence of the antibiotic tetracycline tetR does not bind to its
operators located within the promoter region of the operon and allows transcription. By
combining tetR with the C-terminal domain of VP16 from HSV, known to be essential for the
transcription of the immediate early viral genes, a hybrid transactivator was generated that
stimulates minimal promoters fused to tetracycline operator (tetO) sequences(Gossen and
Bujard 1992). These promoters are virtually silent in the presence of low concentrations of
tetracycline, which prevents the tetracycline-controlled transactivator (tTA) from binding to
tetO sequences. The specificity of the tetR for its operator sequence as well as the high
affinity of tetracycline for tetR and the well-studied chemical and physiological properties of
tetracyclines constitute a basis for an inducible expression system in mammalian cells far
superior to the 1acR/O/IPTG system(Gossen and Bujard 1992). This has already been
demonstrated in plant cells, in which direct repressor action at promoter sites is efficiently
reversed by the antibiotic.

4.6 miRNA
MiRNAs are a newly discovered class of small noncoding RNAs 21-25 nucleotides long that
are involved in a wide variety of developmental processes and may have a role in networking
and fine-tuning gene expression in the cell{Ambros, 2003 #706} {Bartel, 2004 #707}.
MiRNAs are well conserved in eukaryotic organisms, even across species {Bartel, 2004
#707}. Components of the miRNA machinery have been found in archea & eubacteria and
are also believed to be a vital evolutionarily component of genetic regulation. With the latest
human miRNA count swelling to over 800, miRNAs easily account for >3% of all human
genes {Bentwich, 2005 #708}. Under a standard nomenclature system, names are assigned to
experimentally confirmed miRNAs before publication of their discovery. MicroRNAs are
significant phylogenetic markers because of their astonishingly low rate of
evolution(Wheeler, Heimberg et al. 2009). Their origin as a regulatory mechanism stems
18

from previous RNAi (RNA interface) machinery which was initially used as a defense
against exogenous genetic material such as viruses(Pashkovskiy and Ryazansky 2013). This
origin may have permitted the development of morphological innovation, and by making
gene expression more specific and 'fine-tunable', permitted the genesis of complex
organs(Heimberg, Sempere et al. 2008) and perhaps, ultimately, complex life(Peterson,
Dietrich et al. 2009). Indeed, rapid bursts of morphological innovation are generally
associated with a high rate of microRNA accumulation.
Another pathway that uses tiny RNAs as sequence-specific regulators is the RNA
interference pathway. It is said to be an evolutionarily conserved response to the presence of
double stranded RNA in the cell {Meister, 2004 #709}. The dsRNAs are cleaved into 20-base
pair (bp) duplexes of small-interfering RNAs (siRNAs) by Dicer. (Being part of the RNase
III family, Dicer cleaves double-stranded RNA (dsRNA) and pre-microRNA (pre-miRNA)
into short double-stranded RNA fragments called small interfering RNA and micro RNA).
These small RNAs get assembled into multiprotein effector complexes called RNA-induced
silencing complexes (RISC). The siRNAs (small interfering RNAs) then guide the cleavage
of target mRNAs with perfect complementarity. This is important as the finding that some
aspects of biogenesis, protein complexes, and function are shared between siRNAs and
miRNAs has greatly accelerated the study of miRNAs.
While researchers have focused on the study of miRNA expression in physiological and
pathological processes, various technical variables related to microRNA isolation have
emerged. The stability of the stored miRNA samples has often been questioned(Mrz,
Malinova et al. 2009). MicroRNAs are degraded much more easily than mRNAs, partly due
to their length, but also because of the ubiquitously present RNases. This makes it necessary
to cool samples on ice and use RNase-free equipment whenever working with
microRNAs(Liu, Calin et al. 2008).
miRNAs can also be hybridized to microarrays, slides or chips with probes to hundreds or
thousands of miRNA targets, so that relative levels of miRNAs can be determined in different
samples(SHINGARA, KEIGER et al. 2005). MicroRNAs can be both discovered and
profiled by high-throughput sequencing methods (Buermans, Ariyurek et al. 2010). For the
in situ detection of miRNA, LNA(You, Moreira et al. 2006) probes can be used. The
locked conformation of LNA results in enhanced hybridization properties and increases
sensitivity and selectivity, making it ideal for detection of short miRNA.

4.6.1 Biogenesis of miRNA

MiRNAs are transcribed from diverse genomic locations as long primary transcripts (primiRNA) by RNA polymease II {Kim, 2005 #710}. They are embedded in either independent
noncoding RNAs or the introns of protein-coding genes. Additionally, to allow coordinated
expression, some miRNAs are clustered in polycistronic(encodes two or more) transcripts.
Several miRNAs are expressed in a tissue-specific and developmental stage-specific manner.
The expression of the intron-encoded miRNAs could be linked to the transcriptional
regulation of their host gene promoters {Bartel, 2004 #707}. Subsequent to transcription, the
pri-miRNAs are processed by the successive action of two members of the RNase-III family
of enzymes, Drosha and Dicer. In performing their tasks, these proteins are aided by their
companion double-stranded RNA-binding domain-containing proteins. An approximately 70nt(nucleotide) precursor called the pre-miRNA, which can be folded into a stem-loop
structure containing multiple bulges and mismatches, is excised out from the pri-miRNA by
the nuclear Drosha. The pre-miRNA is exported out to the cytoplasm by Exportin-5, in a
Ran-GTP-dependent manner. In the cytoplasm, the pre-miRNA is cleaved by Dicer to
19

generate an approximately 20-bp duplex intermediate {Sontheimer, 2005 #711}. In keeping

with the thermodynamic asymmetry rule, only one strand of the duplex accumulates as the
mature miRNA. The asymmetry rule dictates that the 5 end of the mature miRNA lies at the
end of the duplex which has lower thermodynamic energy {Tomari, 2005 #712}. The mature
miRNA then gets assembled into the effector complexes called miRNPs (miRNAcontaining ribonucleo-protein particles) that share a lot of similarity to the RISC. Functional
RISCs and miRNPs vary in size, ranging from the minimal RISC of ~160 kDa up to the
holo RISC that fractionates at ~80S; human miRNAs were found in complexes of ~15S.
Biochemical characterization of these different complexes has led to the identification of a
number of protein components although the function of these proteins in the RNAi and
miRNA pathways is not clear. However, the only protein consistently found in the RISC and
miRNP complex is the highly conserved Argonaute (Ago) {Carmell, 2002 #713}. The
general consensus is that the minimal effector complex contains a single-stranded small RNA
associated with an Ago protein.
Once the miRNP is assembled, the miRNA guides the complex to its target by base-pairing
with the target mRNA. In plants, miRNAs bind to a single complementary site in either the
coding or 3-untranslated regions (UTRs) of the target mRNA. In contrast, most investigated
animal miRNAs bind to multiple, partially complementary sites in the 3-UTRs. However, the
target sequences inserted into either coding or 5-UTR sequences are also functional
{Kloosterman, 2004 #714}. The complementarity is usually restricted to the nucleotides 28
in the 5 end of the miRNA. These nucleotides form the seed sequence, implying that they
nucleate binding between the miRNA and the target mRNA {Tomari, 2005 #712}. The fate
of the target mRNA is decided by the extent of base-pairing to the miRNA. A miRNA will
direct destruction of the target mRNA if it has perfect or near-perfect complementarity to the
target {Hutvgner, 2002 #715}. On the other hand, the presence of multiple, partially
complementary sites in the target mRNA will direct the inhibition of protein accumulation
without strongly affecting mRNA levels {Bartel, 2004 #707}.

4.6.2 RNA-induced Silencing Complex

The mature miRNA is part of an active RNA-induced silencing complex (RISC) containing
Dicer and many associated proteins {Rana, 2007 #716}. RISC is also known as a microRNA
ribonucleoprotein complex (miRNP); RISC with incorporated miRNA is sometimes referred
to as "miRISC."
Dicer processing of the pre-miRNA is thought to be coupled with unwinding of the duplex.
Generally, only one strand is incorporated into the miRISC, selected on the basis of its
thermodynamic instability and weaker base-pairing relative to the other strand {Krol, 2004
#717}.The position of the stem-loop may also influence strand choice {Lin, 2005 #718}.The
other strand, called the passenger strand due to its lower levels in the steady state, is denoted
with an asterisk (*) and is normally degraded. In some cases, both strands of the duplex are
viable and become functional miRNA that target different mRNA populations {Okamura,
2008 #719}.
Members of the Argonaute (Ago) protein family are central to RISC function. Argonautes are
needed for miRNA-induced silencing and contain two conserved RNA binding domains: a
PAZ domain that can bind the single stranded 3 end of the mature miRNA and a PIWI
domain that structurally resembles ribonuclease-H and functions to interact with the 5 end of
the guide strand. They bind the mature miRNA and orient it for interaction with a target
mRNA. Some argonautes cleave target transcripts directly; argonautes may also recruit
additional proteins to achieve translational repression {Pratt, 2009 #720}. The human
20

genome encodes eight argonaute proteins divided by sequence similarities into two families:
AGO (with four members present in all mammalian cells and called E1F2C/hAgo in
humans), and PIWI (found in the germ line and hematopoietic stem cells) {Pratt, 2009 #720}.

Figure 4.1 :- The miRNA biogenesis pathway. (A) Animal and (B) plant miRNA biogenesis. Mature miRNAs are
indicated in red, whereas the miRNA* strands are in blue. [Courtesy of http://dev.biologists.org]

4.6.3 Function of miRNA

The function of miRNAs appears to be in gene regulation. For that purpose, a miRNA
is complementary to a part of one or more messenger RNAs (mRNAs). Animal miRNAs are
usually complementary to a site in the 3' UTR whereas plant miRNAs are usually
complementary to coding regions of mRNAs {Wang, 2004 #721}. Perfect or near perfect
base pairing with the target RNA promotes cleavage of the RNA {Kawasaki, 2004 #722}.
This is the primary mode of plant miRNAs. In animals miRNAs more often have only partly
the right sequence of nucleotides to bond with the target mRNA. The match-ups are
imperfect. For partially complementary microRNAs to recognise their targets, nucleotides 2
21

7 of the miRNA still have to be perfectly complementary {Maziere, 2007 #723}. Animal
miRNAs inhibit protein translation of the target mRNA {Williams, 2008 #724} (this exists in
plants as well but is less common). MicroRNAs that are partially complementary to a target
can also speed up deadenylation, causing mRNAs to be degraded sooner {Eulalio, 2009
#725}. It is worthwhile to note that whilst degradation of miRNA-targeted mRNA is well
documented, whether or not translational repression is accomplished through mRNA
degradation, translational inhibition, or a combination of the two is hotly debated.
Morozova et al. have found and described nine mechanisms of miRNA action {Morozova,
2012 #726}. They are as follows:

Cap-40S initiation inhibition

60S Ribosomal unit joining inhibition
Elongation inhibition
Ribosome drop-off (premature termination)
Co-translational nascent protein degradation
Sequestration in P-bodies
mRNA Decay (destabilisation)
mRNA Cleavage
Transcriptional inhibition through microRNA-mediated chromatin reorganization
following by gene silencing

4.7 Retina
The retina is a light-sensitive layer of tissue lining the inner surface of the eye. The optics of
the eye create an image of the visual world on the retina (through the cornea and lens), which
serves much the same function as the film in a camera. Light striking the retina initiates a
cascade of chemical and electrical events that ultimately trigger nerve impulses. These are
sent to various visual centres of the brain through the fibres of the optic nerve.
The retina and the optic nerve are considered part of the central nervous system and is
essentially brain tissue. The retina is a multi-layered neural structure whose stratum are
interconnected by synapses. Of the neurons present, the only ones directly sensitive to light
are the photoreceptor cells. These are mainly of two types: the rods and the cones. Rods
chiefly function in dim light and provide black and white vision, while cones sustain daytime
vision and the perception of colour. Another important and rare type of photoreceptor is the
intrinsically sensitive ganglion cell which is important for reflexive responses to bright
daylight.
The vertebrate retina has ten distinct layers. From closest to farthest from the vitreous body that is, from closest to the front exterior of the head towards the interior and back of the head:
Inner limiting membrane basement membrane elaborated by Mller cells(glial cells
found in retina which serve as support for neurons)
Nerve fibre layer axons of the ganglion cell nuclei (note that a thin layer of Mller
cell footplates exists between this layer and the inner limiting membrane)
Ganglion cell layer contains nuclei of ganglion cells, the axons of which become the
optic nerve fibres for messages and some displaced amacrine cells
22

 Inner plexiform layer contains the synapse between the bipolar cell axons and the
dendrites of the ganglion and amacrine cells.
Inner nuclear layer contains the nuclei and surrounding cell bodies (perikarya) of
the amacrine cells, bipolar cells and horizontal cells.
Outer plexiform layer projections of rods and cones ending in the rod spherule and
cone pedicle, respectively. These make synapses with dendrites of bipolar cells. In
the macular region, this is known as the Fiber layer of Henle.
Outer nuclear layer cell bodies of rods and cones
External limiting membrane layer that separates the inner segment portions of the
photoreceptors from their cell nucleus
Photoreceptor layer rods/cones
Retinal pigment epithelium - single layer of cuboidal cells (with extrusions not shown
in diagram)
These can be simplified into 4 main processing stages: photoreception, transmission
to bipolar cells, transmission to ganglion cells which also contain photoreceptors, the
photosensitive ganglion cells, and transmission along the optic nerve. At each synaptic stage
there are also laterally connecting horizontal and amacrine cells.

Figure 4.2 :- Structure of retina[Courtesy of http://www.corpshumain.ca]

23

Retinal Ganglion Cells

A retinal ganglion cell (RGC) is a type of neuron located near the inner surface (the ganglion
cell layer) of the retina of the eye. It receives visual information from photoreceptors via two
intermediate neuron types: horizontal cells and amacrine cells. Retinal ganglion cells
collectively transmit image-forming and non-image forming visual information from the
retina to several regions in the thalamus, hypothalamus, and mesencephalon, or midbrain.
Retinal ganglion cells vary significantly in terms of their size, connections, and responses to
visual stimulation but they all share the defining property of having a long axon that extends
into the brain. These axons form the optic nerve, optic chiasm, and optic tract. A small
percentage of retinal ganglion cells contribute little or nothing to vision, but are themselves
photosensitive; their axons form the retinohypothalamic tract and contribute to circadian
rhythms and pupillary light reflex, the resizing of the pupil. Retinal ganglion cells
spontaneously fire action potentials at a base rate while at rest. Excitation of retinal ganglion
cells results in an increased firing rate while inhibition results in a depressed rate of firing.
There are five main classes of retinal ganglion cells:

Midget retinal ganglion cells(P cells) project to the parvocellular layers of the lateral
geniculate nucleus. These cells are known as midget retinal ganglion cells, based on
the small sizes of their dendritic trees and cell bodies. About 80% of all retinal
ganglion cells are midget cells in the parvocellular pathway. They receive inputs from
relatively few rods and cones. In many cases, they are connected to midget bipolars,
which are linked to one cone each. They have slow conduction velocity, and respond
to changes in color but respond only weakly to changes in contrast unless the change
is great {Kandel, 2000 #727}. They have simple center-surround receptive fields,
where the center may be either ON or OFF while the surround is the opposite.
Parasol retinal ganglion cells (M cells) project to the magnocellular layers of the
lateral geniculate nucleus. These cells are known as parasol retinal ganglion cells,
based on the large sizes of their dendritic trees and cell bodies. About 10% of all
retinal ganglion cells are parasol cells, and these cells are part of the magnocellular
pathway. They receive inputs from relatively many rods and cones. They have fast
conduction velocity, and can respond to low-contrast stimuli, but are not very
sensitive to changes in color {Kandel, 2000 #727}. They have much larger receptive
fields which are nonetheless also center-surround.
Bistratified retinal ganglion cells project to the koniocellular layers of the lateral
geniculate nucleus. Bistratified retinal ganglion cells have been identified only
relatively recently. Koniocellular means cells as small as dust; their small size made
them hard to find. About 10% of all retinal ganglion cells are bistratified cells, and
these cells go through the koniocellular pathway. They receive inputs from
intermediate numbers of rods and cones. They have moderate spatial resolution,
moderate conduction velocity, and can respond to moderate-contrast stimuli. They
may be involved in color vision. They have very large receptive fields that only have

24

centers (no surrounds) and are always ON to the blue cone and OFF to both the red
and green cone.
Photosensitive ganglion cells, including but not limited to the giant retinal ganglion
cells, contain their own photopigment, melanopsin, which makes them respond
directly to light even in the absence of rods and cones. They project to, among other
areas, the suprachiasmatic nucleus (SCN) via the retinohypothalamic tract for setting
and maintaining circadian rhythms. Other retinal ganglion cells projecting to
the lateral geniculate nucleus (LGN) include cells making connections with
the Edinger-Westphal nucleus (EW), for control of the pupillary light reflex, and giant
retinal ganglion cells.
Other ganglion cells projecting to the superior colliculus for eye movements.

25

5. Activity Log
Date

Activity

Observation

28/05/2014

Introduction to lab

Nil

29/05/2014

Understanding about
lab by aiding seniors.

Nil

30/05/2014

Understanding about
lab by aiding seniors.

Nil

02/06/2014

Preparation of
Competent cells
stock

Stock of competent
cells obtained.

03/06/2014

Transformation of
cells with MC23a

After leaving overnight,

colonies were obtained.

04/06/2014

Innoculation of broth

After leaving overnight,

broth filled with MC23a
obtained.

05/06/2014

Isolation of plasmid
and AGE

Plasmid was isolated

and confirmed.

AGE stands for

Agarose Gel
Electrophoresis

06/06/2014

Restriction digestion
of MC23a, pRip 143,
pRip145 and AGE

Gel picture was

obtained clearly and
required pieces were
cut.

Gel pieces were

cut for DNA
retrieval after
comparing to
DNA ladder.

09/06/2014

Retrieval of DNA by
gel elution.

DNA was retrieved.

10/06/2014

Ligation of retrieved
DNA and
transformation

NO COLONIES
OBTAINED

It is hypothesized
that there was an
error in ligation.

11/06/2014

Ligation of retrieved
DNA and
transformation

NO COLONIES
OBTAINED

Troubleshooting
of our actions are
being performed.

12/06/2014

Aiding Seniors

Nil

13/06/2014

Aiding Seniors

Nil

26

Remarks

Viability of cells
must be tested by
transformation

Date

Activity

Observation

16/06/2014

Sick

17/06/2014

Repeat restriction
digestion of MC23a, Required pieces were
PRip143, PRipk5 and cut from Gel and stored
AGE

18/06/2014

Retrieval of DNA by
Gel elution

DNA retrived

19/06/2014

Ligation of retrieved
DNA as MC 143 and
MC 145

DNA was ligated

20/06/2014

Transformation with
MC 143, MC 145

Colonies of MC 143
and MC 145

23/06/2014

Preparation of
MC143, MC 145
stock solution

Stock of solutions
prepared and kept

24/06/2014

Isolation of Plasmid

Plasmid was isolated

25/06/2014

Digestion of plasmid
and AGE

Remarks

We began this
with new stock of
inserts.

This was done by

inoculating broth
and storing it in
glycerol.

ERROR IN LOADING

Hand shook while

loading causing
spillage.

26/06/2014

Digestion of
plasmids and AGE

ERROR IN LOADING

Hand moved gel

causing a sliding
in image.

27/06/2014

Digestion and AGE

Picture not clear

Plasmid has run

out.

30/06/2014

Innoculation of
MC143 from stock
MC 145

Broths of MC 143, MC
145 obtained

01/07/2014

Inoculation repeat,
Isolation of plasmid

No Plasmid

02/07/2014

Isolation of plasmid

Plasmid obtained

03/07/2014

Restriction Digestion

NOT ENOUGH
27

Plasmid got
washed out with
alcohol.

Amount of

Date

Activity

Observation

Remarks

and AGE

PLASMID

plasmid was not

enough.

04/07/2014

Restriction digestion
and AGE

Plasmid obtained and

vector insert verified

07/07/2014

Sequencing PCR and

PCR cleanup

Final solution obtained

Dilution of
plasmid was
(1:15)

ERROR

The sequence did

not come
correctly.
Dilution of
plasmid changed
to (1:10)

08/07/2014

Sequencing Main

09/07/2014

Sequencing PCR and

PCR cleanup

Final solution obtained

10/07/2014

Sequencing Main

Plasmid verified

11/07/2014

Transfection of
plasmid MC143 and
MC145

ERROR

14/07/2014

Review of draft
report

Citations to be
improved

15/07/2014

Review of final
report

28

Could not be
completed as
plasmid not high
enough quality.

6. EXPERIMENTS PERFORMED AT RGCB

6.1 PREPARATION OF COMPETENT E. COLI CELLS USING CaCl2

Prepare Solutions
1. Luria-Bertani (LB) media (1 L):
Mix 10 g of Bacto-tryptone, 5 of Yeast extract, and 10 g of NaCl (for taste). pH to 7.5 w/
NaOH. And dH2O to 1 L (Autoclave)
2. 1M CaCl2 (1 L):
Mix 111 g of CaCl2 (anhydrous) and 1 L of dH2O. Filter sterilize through a 0.22m filter
3. 0.1M CaCl2 (1 L):
Mix 100 mL of 1M CaCl2 with 900 mL of dH2O. Filter sterilize through a 0.22m filter
4. 50% Glycerol (500 mL):
Mix 50 mL of Glycerol with 50 mL of dH2O (Autoclave)
5. 0.1M CaCl2 + 15% glycerol:
Mix 100 mL of 1M CaCl2, 300 mL of 50% Glycerol, and 600 mL of dH2O
6. LB plates:
Mix 500 mL of LB media with 7 g of Agar (Autoclave). Cool to ~55-65oC prior to pouring.
The addition of antibiotics should be made before pouring and at a temperature not higher
than 55oC. Antibiotics can also be spread on previously made plates, but this is not very
effective (unequal absorption, etc...)
PROCEDURE
1. Streak E.coli cells (DH5a, HB101, GM8) on an LB plate; (BL21(DE3)LysS cells on LB
plate+34 mg/ml chloramphenicol)
2. Allow cells to grow at 37oC overnight
3. Place one colony in 10 mL LB media (+antibiotic selection if necessary), grow overnight
at 37oC
4. Take 2 ml LB media and save for blank. Transfer 5 mL overnight DH5a culture into
500mL LB media in 3 L flask
5. Allow cell to grow at 37oC (250 rpm), until OD600= 0.4 (~2-3 hours)
6. Transfer cells to 2 centrifuge bottles (250 mL), and place cells on ice for 20 mins
7. Centrifuge cells in Sorval GSA rotor at 4oC for 10 mins at 3,000 g Subsequent
resuspensions may be done in the same bottle.
Cells must remain cold for the rest of the procedure: Transport tubes on ice and resuspend on
ice in the cold room
8. Pour off media and resuspend cells in 30 mL of cold 0.1 M CaCl2. Transfer the suspended
cells into 50 mL polypropylene falcon tubes, and incubate on ice for 30 mins
9. Centrifuge cells using Sorval RT6000B rotor at 4oC for 10 mins at 3,000 g (2500 rpm)
10. Pour supernatant and resuspend cells (by pipetting) in 8 mL cold 0.1M CaCl2 containing
15% glycerol. Transfer 140 mL into (1.5 mL) Ependorff tubes placed on ice. Freeze the cells
in liquid nitrogen. Cells stored at -80oC can be used for transformation for up to ~6 months
NOTE: through the process, cells should be treated with care. No vortexing or excess
pipetting should be performed, especially when the cells have been resuspended in CaCl2
because lysis will result, decreasing the amount of competent cells). Also, depending on the

29

density of the cells, higher or lower volumes CaCl2 can be used to increase the concentration
of cells per tube.

6.2 Transforming Competent Cells with Plasmid DNA

Summary:
Transformation - the heritable modification of the properties of a competent bacterium by
DNA from another bacterial strain.
DH5 competent cells will be used (e.g., PGC Scientific). A plasmid with DNA
resistant to ampicillin from another bacterial strain (foreign DNA) will be introduced to the
DNA of the competent bacterium (host DNA) and streaked on LB agar plates with antibiotic.
Cells with the foreign DNA will be resistant to ampicillin, resulting in colonies on LB agar
with ampicillin. Cells without the foreign DNA will lyse!!

Procedure:
1. Remove cells from 70oC and let thaw on ice.
WARNING: When handling frozen competent cells, it is important to KEEP IT COOL!
Frozen cells are very sensitive to being warm and will not work as well if not kept on ice
as much as possible.
2. Place the cells on benchtop to thaw. Replace on ice.
3. After mixing gently, aliquot ~25 L of cells into chilled, 17x100 mm polypropylene
tube(s), e.g., Falcon 2059. Note: 25 L is enough for Transformation while 50 L is
required for Ligation.
4. Add 1.25 L or less plasmid DNA (the volume of DNA solution should not exceed 5% of
the volume of the competent cells or no more than 50 ng in a volume of 10 L or less).
You dont need very much plasmid to get transformed colonies. Generally around 20 ng
(nanograms) is enough.
5. Gently swirl tube(s) for a few seconds to mix by finger flicking tube. Note: If a control
is desired, repeat this step with 1-2 L (10-20 ng) of pUC19 in a separate tube.
6. Then incubate tube on ice for 30 minutes.
7. Heat shock tube(s) by placing in 42oC water bath for ~45 seconds without shaking.
8. Replace tube(s) on ice for 2-5 minutes.
9. Add 900 L of S.O.C. (2% Tryptone, 0.5% Yeast Extract, 0.4% glucose, 10 mM NaCl,
2.5 mM KCl, 10 mM MgCl2, & MgSO4).
10. Gently shake and incubate tube(s) at ~200 rpm for 30-60 minutes at 37oC.
11. Spread on LB agar plates containing appropriate antibiotic (e.g., 100 g/mL ampicillin
for control pUC19). Tip: Spread by evenly spreading cells over the plate using a
hockey puck spreader. Be sure that you have dipped your spreader in ethanol and
flamed it before spreading.
30

12. Place in the 37oC incubator with the agar side up and the lid side down. Incubate
overnight and remove to fridge (-4oC) sometime the next day.
Reagents/Solutions:
1. SOC Medium (1 Liter)
800 mL of deionized H2O
20 g Bacto-Tryptone
5 g Bacto-Yeast Extract
0.5 g NaCl
Shake until the solutes have dissolved. Add 10 mL of a 250 mM solution of KCl. (This
solution is made by dissolving 1.86 g of KCl in 100 mL of deionized H2O.) adjust pH to
7.0 with 5 N NaOH (~0.2 mL). Adjust the volume of the solution to 1 Liter with
deionized H2O. Autoclave.
Just before use, add 5 mL of a sterile solution of 2 M MgCl2. (This solution is made by
dissolving 19 g of MgCl2 in 90 mL of deionized H2O. Adjust the volume of the solution
to 100 mL with deionized H2O. Autoclave).
*20 mM glucose (After SOC has been autoclaved, allow to cool to 60oC or less and then
add 20 mL of a sterile 1 M solution glucose. This solution is made by dissolving 18 g of
glucose in 90 mL of deionized H2O. after the sugar has dissolved, adjust volume of the
solution to 100 mL with deionized H2O and sterilize by filtration through a 0.22-micron
filter.)
2. 3 M NaOAc pH 5.2 (1 Liter)
If FW=82.03 g/mol, add:
246.1 g CH3COONa (anhydrous)
800 mL deionized H2O
pH to 5.2 with Glacial Acetic Acid
Bring to volume of 1 Liter
Autoclave

3. LB agar plates (1 Liter)

31

800 mL d H2O
10 g Bacto-Tryptone
5 g Bacto-Yeast extract
10 g NaCl
pH to 7.0
Bring to volume of 1 Liter
*Autoclave

*Just before autoclaving, add 15 g of Bacto-agar solidifying agent/1 Liter LB Medium

Useful Tables:

Antibiotic Solutions
Working concentration
Stock solution
concentrations

Ampicillin

Kanamycin

in ethanol

60g/mL

-20oC

20 g/mL

60g/mL

-20oC

25 g/mL

170g/mL

-20oC

10 g/mL

50g/mL

-20oC

10 g/mL

50g/mL

-20oC

10 g/mL

50g/mL

10 mg/ml

10 mg/ml
in H20

Tetracycline

20 g/mL

34 mg/ml

in H20
Streptomycin

-20oC

storage

50 mg/ml
in H20

Chloramphenicol

Relaxed
plasmids

50 mg/ml
in H20

Carbenicillin

Stringent
plasmids

50 mg/ml
in ethanol

32

aStock solutions of antibiotics dissolved in H2O should be sterilized by filtration through a

0.22-micron filter. Antibiotics dissolved in ethanol need not be sterilized. Store solution in
light-tight containers.
bMagnesium ions are antagonists of tetracycline. Use media without magnesium salts (e.g.,
LB Medium) for selection of bacteria resistant to tetracycline.
Media Containing Agar or Agarose
Prepare liquid media according to the recipes given above. Just before autoclaving, add one
of the following:
Bacto-agar

(for plates) 15 g/Liter

Bacto-agar

(for top agar) 7 g/Liter

Agarose

(for plates) 15 g/Liter

Agarose

(for top agar) 7 g/Liter

6.3 Plasmid Isolation Using Alkaline Lysis

1. 5 ml LB medium containing proper antibiotics were inoculated with a single bacterial
colony. The tube was incubated at 37 C overnight with vigorous shaking at 360 rpm.
2. Pellet bacteria from the culture at 10,000 x g for 5 minutes at room temperature.
3. Discard the supernatant.
4. Resuspend bacterial pellet in a total of 1 ml ice-cooled solution I (50 mM). Pipette up and
down or vortex as necessary to fully resuspend the bacteria.
5. Add 2 ml room temperature 0.2 N NaOH/1.0% SDS to the suspension. Mix thoroughly by
repeated gentle inversion. Do not vortex.
6. Add 1.5 ml ice-cold Solution III to the lysate. Mix thoroughly by repeated gentle inversion.
Do not vortex.
Incubate on ice for 5 minutes.
7. Centrifuge at 15,500 x g for 10 minutes.
8. Recover resulting supernatant and add 7microlitres of 20mg/l RNAse.
9. Add 400 microlitres chloroform. Mix by inversion and centrifuge for 2minutes before
recovering upper aqueous phase. Repeat the procedure.

33

Add equal volume isopropanol to precipitate the plasmid DNA. Mix thoroughly by repeated
gentle inversion. Do not vortex. Incubate for 10-15 minutes at room temperature.
10. Centrifuge at 15,500 x g for 15 minutes.
11. Removal of resulting supernatant. The pellet is plasmid DNA.
12. Rinse the pellet in ice-cold 70% EtOH and air-dry for about 10 minutes to allow the
EtOH to evaporate.
13. Add ddH2O or TE to dissolve the pellet. After addition of 2ul RNase A (10mg/ml), the
mixture was incubated for 20 minutes at room temperature to remove RNA.
Note:
1. Spin down your cells. Your DNA is still in the cells, so it is in the pellet at this stage.
2. Discard the supernatant and to even invert the tube and wipe the lip with a Kim-wipe or Qtip.
3. Resuspend the cells in buffer (often Tris) and EDTA. EDTA chelates divalent metals
(primarily magnesium and calcium). Removal of these cations destabilizes the cell
membrane. It also inhibits DNases. Glucose should also be added to maintain osmolarity and
prevent the buffer from bursting the cells.
14. Lyse the cells with sodium hydroxide (NaOH) and SDS. This highly alkaline solution
gave rise to the name of this technique. Mix this by gentle inversion and incubate on ice for
five minutes (but no longer, or your DNA will be irreversibly denatured). Three things
happen during this stage:
a. SDS pops holes in the cell membranes. SDS (sodium dodecyl (lauryl) sulfate) is a
detergent found in many common items such as soap, shampoo and toothpaste.
b. NaOH loosens the cell walls and releases the plasmid DNA and sheared cellular DNA.
c. NaOH denatures the DNA. Cellular DNA becomes linearized and the strands are separated.
Plasmid DNA is circular and remains topologically constrained.
15. Renature the plasmid DNA and get rid of the garbage. Add potassium acetate (KAc),
which does three things:
a. Circular DNA is allowed to renature. Sheared cellular DNA remains denatured as single
stranded DNA (ssDNA).
b. The ssDNA is precipitated, since large ssDNA molecules are insoluble in high salt.
c. Adding sodium acetate to the SDS forms KDS, which is insoluble. This will allow for the
easy removal of the SDS from your plasmid DNA.

34

Now that you've made it easy to separate many of the contaminants, centrifuge to remove cell
debris, KDS and cellular ssDNA. Your plasmid DNA is in the supernatant, while all of the
garbage is in the pellet.
16. Precipitate the plasmid DNA by alcohol precipitation (ethanol or isopropanol) and a salt
(such as ammonium acetate, lithium chloride, sodium chloride or sodium acetate) and spin
this down. DNA is negatively charged, so adding a salt masks the charges and allows DNA to
precipitate. This will place your DNA in the pellet.
17. Rinse the pelletyour plasmid DNAin ice-cold 70% EtOH and air-dry for about 10
minutes to allow the EtOH to evaporate.
18. Resuspend your now clean DNA pellet in buffer (often Tris) and EDTA plus RNases to
cleave any remaining RNA. Your DNA is now back in solution. DNA of this purity is good
for a number of uses, such as in vitro transcription or translation or cutting with some
enzymes. If you are sequencing or transforming thisDNA into mammalian cells, you'll want
to use additional purification techniques such as phenol extraction, Qiagen column
purification, or silica-based purification.
Isolation of the Plasmid after Alkaline Lysis
The plasmid "miniprep " method is useful for preparing partially purified plasmid DNA in
small quantities from a number of transformants. It relies on an alkaline SDS lysis to free the
plasmid DNA from the cell, leaving behind the E. coli chromosomal DNA with cell wall
debris. The protocol described involves three basic steps: growth of bacteria and
amplification of the plasmid; harvesting and lysis of the bacteria; and purification of the
plasmid DNA.
These purification procedures exploit in one way or another the two major differences
between Escherichia coli DNA and plasmid DNA:
1. The E. coli chromosome is much larger than the DNA of plasmids used as vectors.
2. The bulk of E. coli DNA extracted from cells is obtained as broken, linear molecules. By
contrast, most plasmid DNA is extracted in a covalently closed, circular form.
The purification protocol therefore involves a differential precipitation step, in which the long
strands of E. coli DNA, entangled in the remnants of lysed cells, are preferentially removed.
Because each of the complementary strands of plasmid DNA is a covalently closed circle, the
strands cannot be separated (without breaking one of them) by conditions such as exposure to
mild alkali (up to pH 12.5), which break most of the hydrogen bonds of DNA. Closed
circular molecules regain their native configuration when returned to neutral pH.E. coli
remains in the denatured state. This method provides enough purified plasmid DNA for
sequencing.
5 ml LB medium were inoculated with a single bacterial colony. The tube was incubated at
37psy176 C overnight with vigorous shaking. 4.5 ml of the culture were centrifuged for 20
35

minutes at 3500 rpm at 4psy176 C. The remainder of the overnight culture was stored at
4psy176 C. The medium was removed, leaving the bacteria pellet as dry as possible. The
pellet was resuspended in 150 l ice-cold Lysis buffer I (50 mM glucose, 10 mM EDTA, 25
mM Tris, pH 8.0) and stored for 5 minutes at room temperature. After adding 300 l freshly
prepared and mixing by inversion, the mixture was incubated for 5 minutes on ice. Now 225 l
ice-cold 3M KAc/5M HAc (pH 6.0) were added and mixed gently. The tube was stored on
ice for 5 minutes, precipitating the chromosomal bacteria DNA. After centrifugation for 15
minutes at 13000 rpm at 4psy176 C, the supernatant was transferred to a fresh tube. Proteins
were removed by vortexing with 400 l phenol, adding 300 l Sevac and centrifuging for 2
minutes at 13000 rpm.
500-600 l of the aqueous layer were removed and mixed with 1 ml ethanol to precipitate the
DNA. After incubating for 5 minutes at room temperature, the Eppendorf tube was
centrifuged for 10 minutes at 13000 rpm at 4psy176 C. The supernatant was removed, the
pellet washed with 70% ethanol and recentrifuged. After vacuum drying,
Solution I (Lysis buffer I): 50 mM glucose, 10 mM EDTA, 25 mM Tris, pH 8.0. Store at 0C
a. 10ml 500mM Glucose
b. 2ml 500mM EDTA pH 8.0
c. 2.5ml 1M Tris pH 8.0
d. 85.5ml H2O
e. Autoclave and store at 4C
Solution II (Lysis buffer II): Freshly prepared 0.2 N NaOH, 1% SDS. Store at room
temperature (RT)
Isopropanol: Stored at -20 0C
Solution III (Lysis buffer III): 3M KOAc, pH 6.0
a. 60ml 5M potassium acetate (49.07g potassium acetate in 100ml H2O)
b. 11.5ml glacial acetate
c. 28.5ml H2O

36

6.4 AGAROSE GEL ELECTROPHORESIS

Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins.
Most every molecular biology research laboratory routinely uses agarose gel electrophoresis
for the preparation and analysis of DNA. We will be using agarose gel electrophoresis to
determine the presence and size of PCR products.

Background:
Electrophoresis is a method of separating substances based on the rate of movement while
under the influence of an electric field. Agarose is a polysaccharide purified from seaweed.
An agarose gel is created by suspending dry agarose in a buffer solution, boiling until the
solution becomes clear, and then pouring it into a casting tray and allowing it to cool. The
result is a flexible gelatin-like slab. During electrophoresis, the gel is submersed in a
chamber containing a buffer solution and a positive and negative electrode. The DNA to be
analyzed is forced through the pores of the gel by the electrical current. Under an electrical
field, DNA will move to the positive electrode (red) and away from the negative electrode
(black). Several factors influence how fast the DNA moves, including; the strength of the
electrical field, the concentration of agarose in the gel and most importantly, the size of the
DNA molecules. Smaller DNA molecules move through the agarose faster than larger
molecules. DNA itself is not visible within an agarose gel. The DNA will be visualized by
the use of a dye that binds to DNA.
Purpose: To determine the presence or absence of PCR products and quantify the size
(length of the DNA molecule) of the product.
Materials needed:

Agarose
TAE Buffer
6X Sample Loading Buffer
DNA ladder standard
Electrophoresis chamber
Power supply
Gel casting tray and combs
DNA stain
Staining tray
Gloves
Pipette and tips

37

Recipes:

TAE Buffer
4.84 g Tris Base
1.14 ml Glacial Acetic Acid
2 ml 0.5M EDTA (pH 8.0)
- bring the total volume up to 1L with water

Add Tris base to ~900 ml H2O. Add acetic acid and EDTA to solution and mix. Pour mixture
into 1 L graduated cylinder and add H2O to a total volume of 1 L.
Note for convenience a concentrated stock of TAE buffer (either 10X or 50X) is often made ahead of
time and diluted with water to 1X concentration prior to use.

6X Sample Loading Buffer

1 ml sterile H2O
1 ml Glycerol
enough bromophenol blue to make the buffer deep blue (~ 0.05 mg)
-for long term storage, keep sample loading buffer frozen.
QUIKView DNA Stain
25 ml WARDS QUIKView DNA Stain
475 ml warm water (50-55 C)

Agarose Gel Electrophoresis Protocol

Preparing the agarose gel
Measure 1.25 g Agarose powder and add it to a 500 ml flask
Add 125 ml TAE Buffer to the flask. (the total gel volume well vary depending on the size
of the casting tray)
Melt the agarose in a microwave or hot water bath until the solution becomes clear. (if
using a microwave, heat the solution for several short intervals - do not let the solution boil
for long periods as it may boil out of the flask).
Let the solution cool to about 50-55C, swirling the flask occasionally to cool evenly.
Seal the ends of the casting tray with two layers of tape.
Place the combs in the gel casting tray.

38

 Pour the melted agarose solution into the casting tray and let cool until it is solid (it should
appear milky white).
Carefully pull out the combs and remove the tape.
Place the gel in the electrophoresis chamber.
Add enough TAE Buffer so that there is about 2-3 mm of buffer over the gel.
Note gels can be made several days prior to use and sealed in plastic wrap (without
combs). If the gel becomes excessively dry, allow it to rehydrate in the buffer within the gel
box for a few minutes prior to loading samples.

Loading the gel

Add 6 l of 6X Sample Loading Buffer to each 25 l PCR reaction
Record the order each sample will be loaded on the gel, including who prepared the
sample, the DNA template - what organism the DNA came from, controls and ladder.
Carefully pipette 20 l of each sample/Sample Loading Buffer mixture into separate wells
in the gel.
Pipette 10 l of the DNA ladder standard into at least one well of each row on the gel.
Note if you are running multiple gels, avoid later confusion by loading the DNA ladder in
different lanes on each gel.

6.5 RESTRICTION DIGESTION

In the micro centrifuge tube prepare the following reaction mixture :
Plasmid
Bam H1 10 X Buffer
Bam H1
Hind III
Water
Total

4 l
2 l
0.5 l
0.5l
13 l
20 l

After this, incubate at 37C for 2 hours

39

6.6 LIGATION
Prepare two micro centrifuge tubes with following mixtures:

pRip 143 (Insert 1)

MC23a (Vector)
T4 DNA ligase
T4 DNA ligase buffer
Total

pRip 145 (Insert 1)

MC23a (Vector)
T4 DNA ligase
T4 DNA ligase buffer
Total

16 l
1 l
1 l
2 l
20 l

16 l
1 l
1 l
2 l
20 l

Incubate at 16C overnight (minimum 2 hours)

6.7 SEQUENCING
In the microcentrifuge tube, prepare the following for MC143 FW, MC143 RV, MC145 FW
and MC145 RV.
AB1 mix
(1:10) plasmid
dd H2O
FW / RV primer
Total

1 l
3 l
5.5 l
0.5l
10 l

Run it in the corresponding PCR program

10 l PCR reaction + 10 l ddH2O + 2 l 125 mM EDTA
Vortex and incubate at room temperature for 15 minutes after vortexing
Spin at max speed for 20 minutes at room temperature
Wash with 75- 80% ethanol twice for 20 minutes each at room temperature
Keep for drying by lid open preferably in dark
Next day place it for sequencing

40

7. Conclusion
We have taken a constructed pRIPM vector with pre-miR-143 / pre-miR-145. From this
we cloned the amplified miRNA sequence onto a new MC vector to obtain MC143 and
MC145. We can now use this vector in order to complete a tetracycline inducible
expression system. Then, we can expose the system to doxycycline and tetracycline. The
binding strength of miRNAs in 3UTR can be confirmed by luciferase activity. Thus the
Tet-On expression system can be used for the application of studies in miRNA
interactions in retina.
The Above studies we successfully developed:
Cloning of pre-miR-143 and pre-miR-145 genes downstream of mCherry gene in
pmRi-mCherry basic vector.

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