Download GENERATION OF BANK POST-TRANSCRIPTIONAL FUSIONS OF

GENERATION OF BANK POST-TRANSCRIPTIONAL FUSIONS OF Azotobacter
vinelandii.
Research Center of Microbiological Sciences ICUAP-BUAP
Microbial Molecular Genetics Department.
Project Director: Dr. Miguel Castañeda Lucio. Project Advisor: Mtra. Silvia Ma. Del
Carmen García García. Rojas Morales Emmanuel.
Abstract.
The production of Alginate and PHB (Poly-β-hydroxybutyrate) are produces by
more microorganisms how Azotobacter vinelandii, that is a bacterium gram-negative,
pleomorphic it can be two forms, dyads or cysts. The two metabolites are of importance
industry, with PHB can be made surgical suture threads, as they are biodegradable in the
human body. And whit alginate is used to molding pastes can be made for your teeth
because it is viscosifying agent and some cosmetics. The importance of generate a bank of
genes is made a stock of information of genes that produced the two metabolites. The
Azotobacter vinelandii has been described in diverse works, for its wide capacity to
synthesize alginate and synthesize down quality of PHB. For this reason in this work
mutants of Azotovacter vinelandii affected in the accumulation of Alginate, through insert
of the transposon mini Tn5 were obtained.
Keywords: PHB (Poly-β-hydroxybutyrate), pUTmini Tn 5 (Transposone), Kmr
(Kanamicine resistence), Smr (Estreptemicene resistence), SPCr (Espectinemicine
resistence), Triparental conjugation, E. coli DH5, plasmid R6K.
alginate. Thanks to their properties, the alginate
is used as a thickening agent and viscosifier. It
has a wide variety of applications in industries
such
as
food,
pharmaceutical
and
biotechnology industries. However, the large
scale production of alginate is made from
extracts of wild seaweed, and is too expensive,
so it is of great interest to study
microorganisms such as Azotobacter vinelandii,
which in future will be used on an industrial
scale for production of these products.
INTRODUCTION
In the laboratory of molecular microbial
genetics is modeled to study the bacterium
Azotobacter vinelandii (A. vinelandii is a
proteobacterium that belongs to the family
Pseudomonaceae.
It
is
gram-negative,
pleomorphic, and can be present individually or
in dyads is movable by peritrichous flagella
under adverse environmental conditions
undergoes a process of differentiation to form
desiccation-resistant structures, which are
known as cysts.
BMM in the lab examines the ways in which
these metabolites are produced and regulated.
The production of both metabolites is
controlled by a dual signaling system
component called Gacs / A, which consists of a
kinase histidinica (Gacs) located in the cell
membrane that upon receiving the appropriate
stimulus is capable of self-phosphorylated and
subsequently phosphorylate a response
A. vinelandii has characteristics that are under
study, as the fixing of nitrogen in the presence
of oxygen and the versatility of your
metabolism, it is capable of producing
secondary metabolites of industrial interest: the
intracellular polyester poly-β-hydroxybutyrate
(PHB) and extracellular polysaccharide
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regulator (GacA), which is a protein with
DNA-binding motifs which acts as a
transcriptional regulator of the metabolic
pathways that produce alginate and PHB, in
addition to controlling certain other control
systems, such as post-regulator RSMA
transcriptional regulator and the small RNA
RSMB. In addition to alginate and PHB, the
regulatory cascade controlling other genes
might be involved in the synthesis of other
metabolites and physiological processes.
Chapter 10 pages 278-297). Are widely used in
cloning and mutagenesis experiments.
It is important to create tools to facilitate the
work in the laboratory, using a bank of mutants
will develop a series of strategies for evaluating
new phenotypes and the regulation of
expression of genes related to Gacs / A-Rsm.
The transposon Tn5 containing the gene for
resistance to an aminoglycoside antibiotic
which can be expressed in a wide variety of
prokaryotes
and
eukaryotes.
Several
transposons have been integrated into plasmids
(eg., The Tn1 and Tn3) or chromosomes (eg.,
The Tn5) (Lorenzo and Timmis 1990).
Transposons are therefore for a wide variety of
purposes in gene technology. By transposon
mutagenesis offers a variety of advantages.
Mutagenesis by transposon
Transposon mutagenesis is another method for
introducing
mutations
via
insertion.
Transposons are known in both prokaryotes
and eukaryotes and can be inserted at arbitrary
locations in the genome. (Gabriela Levitus,
Chapter 4, pages 53-54).
A bank of mutants can be developed by use of
transposons. A transposon is a mobile genetic
element that can move from a genomic location
to another, thanks to the presence of short
repeated sequences that flank and which is
capable of replicating and inserting a copy in a
new location in the genome. These encode all
the enzymes necessary for insertion.
Mutant phenotype can be obtained with a very
low rate of reversal. The integration of a
transposon causes an interruption in the
transcript, so that the transposon mutagenesis
presents a polar effect (Maniatis, T., EF Fritsch,
and J. Sambrook. 1982). Because of this the
site in which the transposon is integrated in the
operon can be readily determined by an assay
of the activity of an enzyme or by measuring
the accumulation of an intermediate product
(Augustine Lopez, Lopez-Munguia page 443).
BACKGROUND
Transposons
They have developed a series of transposon
mutagenesis to perform in the laboratory the
most used are the mini-transposons as they
have many advantages, are easy to use, possess
variants with resistance genes. (The most cited
in the literature are the mini Tn5 and Tn10 mini
Lorenzo and Timmis 1990).
Insertion mutagenesis with pUTmini-Tn5
The mini-transposon mini-Tn5 transposon is
derived from Tn5 compound. Each of these
elements lack the gene coding for a transposase
gene and include, which confers resistance to
an antibiotic, flanked by a sequence of 19 bp
from the ends of the I and O Tn5. In our case
we used the mini-Tn5 Sm containing the gene
that gives resistance to Sm. (Way et al., 1984).
The mini Tn5 transposons are mini-transposons
that are transferred using a suicide plasmid
origin of replication R6K (Lorenzo and Timmis
1990) and offer a selection of markers with
various antibiotics such as Streptomycin /
Spectinomycin, Kanamycin, Streptomycin,
Tetracycline, Chloramphenicol . It has been
determined showing a transposition frequency
of 10-5 to 10-6, but may vary with different
bacterial species (Michael T. Jack Parker
The transmission vector, plasmid pUT (de
Lorenzo et al., 1990) has a replication origin πdependent protein (derived from plasmid R6K)
2
and the transfer origin derived from plasmid
autoconjugativo oriT RP4. Furthermore the
plasmid pUT has the gene that encodes
transposase needed for transposition of the
mini-Tn5.
R6K (π-dependent protein), carries the
transposase gene tnp cis and whose conjugal
transfer to recipient cells is mediated by RP4
mobilization functions in the donor (Michael T.
Jack Parker Chapter 10 pages 278-297)
The pUTmini-Tn5 is stable and capable of
being mobilized, as in strain S17.1 pir , this
strain produces a protein expressed π and
functions for the conjugation of RP4.
Mutagenesis on the plasmid is transferred by
conjugation to a non-producing strain of the
protein and thus can not maintain the plasmid.
There are many cases of conjugative plasmids,
but some of them can be transferred to recipient
strains, when "living" in the donor cell with
another capable of mobilizing conjugative
plasmid. This is usually due to these plasmids
mobilized autotransmisibles but lack the tra
region but have a region called the mob (the
"mobilizable"), which is recognized by Tra
conjugation proteins supplied in trans by the
plasmid mobilizing, although the region
mobilizable plasmid mob region provides a cis
(sequence oriT) and a pair of functions in trans
(specific endonuclease which acts on oriT, and
a helicase) (Charles Round, Guillermina
Alonso September 10, 2007).
The conjugants selected by antibiotic resistance
encoded by the mini-transposon mutants are
mini-Tn5 insertion. The advantage is the use of
mini-transposon is that having no transposase
encoded generate stable insertions. Also in the
case of mini-Tn5 Sm has no transcription
terminator sequences flanking the gene for
streptomycin, which reduces polar effects.
For the plasmid can be mobilized pUTmini Tn5
plasmids does so through so-called "helper
plasmid" that may force him Mob + plasmid
work to mobilize (Guadalupe Espin 2006). The
strain of E. coli DH5 pir containing the pir gene
and is designed for cloning and propagating
plasmids with R6K replication origin like
pUTmini Tn5. To transfer these plasmids
directly from a donor strain DH5 pir to a
recipient strain is necessary to carry out a
triparenteral conjugation with a helper strain
such as E. coli DH5a pRK2013 (Guadalupe
Espin 2006).
The procedure followed for the mutagenesis is
that described by Lawrence et al., 1990. Mini
Tn5 transposons are constructed in a manner to
simplify the generation of substantial insertion
mutants, fusions in vivo as reporter genes are
the gusA gene or Lac Z and the introduction of
foreign DNA fragments in the chromosome of
a wide variety of bacteria including gramnegative enteric bacteria and soil such as
Pseudomonas species.
The mini-transposon consisting of genes
specifying resistance to an antibiotic
(kanamycin, streptomycin, chloramphenicol,
tetracycline), as selection markers and a unique
NotI cloning site, flanked by 19 bp repeat
terminal Tn5. Other derivatives of Tn5 also
contain lacZ genes. PhoA, lux AB, or xylE
lacking its native promoter located after the
terminal repeats in an orientation that provides
the generation of gene fusions operons
(Maniatis, T., EF Fritsch, and J. Sambrook.
1982).
The bank will be constructed transcriptional
fusions using the gusA reporter gene encoding
β-glucuronidase for molecular biology, the βglucuronidase is used as a reporter gene to
control gene expression in mammalian cells,
plants, and microorganisms (Carlos Pena and
Enrique Galindo 2001). Its monitoring is
performed by using a GUS assay which allows
determination of the expression of the gene in
question (iván darío barrero-Farfán February
11, 2008).
Transposons are located on a suicide plasmid
which carries a replication origin from plasmid
3
The bank will be constructed transcriptional
fusions introducing a gusA reporter gene into
the NotI site of plasmid pUTmini Tn5, which
will carry out the transcriptional fusion with the
reporter gene under the control of a gene
upstream of it. Because the reporter gene has its
own sites of initiation of translation, as the
reporter gene product is not regulated
translationally, the amount of reporter gene
product is proportional to the transcription of
the gene upstream of the reporter. The
translation of the mutated gene stops before the
reporter gene, and translation restart at sites of
translation initiation in front of the reporter
gene (Primrose et al. 2001). Therefore, as a
result there is the product of two genes. The
truncated protein and reporter protein.
OBJECTIVES
GENERAL PURPOSE
Build a bench transcriptional fusions.
SPECIFIC OBJECTIVES
1. Establish conditions for obtaining a
significant number of mutant clones
enough to have represented the genome
of Azotobacter vinelandii.
2. Test the conditions of a massive
mutagenesis experiment.
MATERIALS AND METHODS
Conjugation triparenteral
The method of conjugation triparenteral for
transformation of strain E, Azotobacter
vinelandii, using the plasmids from E. pUTmini
coli Tn5 Km-resistant and Sm and the helper
plasmid pRK2013 kmr.
When performing the construction of genomic
libraries is important to obtain sufficient
numbers of clones as this will be represented in
these whole genome to be studied, in this case
the genome of Azotobacter vinelandii is about
5.300 Kb so to have represented all of its
genome in a bank require an approximate
amount of 5000-6000 colonies each with
insertions in different fragments (Guadalupe
Espin March 6, 2009).
In practice, the size of the library can be
calculated simply on the basis of the probability
that a particular sequence to be represented in
the library (Brown 2001). There is a formula
that takes into account certain factors and
results in the "number of clones" that we
should consider to have represented the
genome that interests us, the formula is:
Strain of A. vinelandii is a mucosal
morphology in the colonies formed.
For conjugation is started with a culture of A.
vinelandii in a 250 ml Erlenmeyer flask with 50
ml of nalidixic acid BS more liquid medium
was inoculated with two roast A. vinelandii
from a fresh plate, was incubated with stirring
for 24 hours at 30oC.
N = ln (1 - P) / ln (1 - a / b)
Where N is the number of clones required, P is
the probability that a particular sequence
desired to be represented (typically set at 0.95
or 0.99), a is the average size of DNA
fragments to be cloned, b is the genome size
(expressed in the same units as a). Using this
formula, we can determine the magnitude of
the task and plan a strategy for cloning.
To prepare a culture of strains of E. coli
(pUTminiTn5 Smr and the helper pRK2013
Kmr) in 15 ml falcon tubes with 5 ml liquid LB
medium plus its respective antibiotic Sm and
Km, was incubated for 12 hours at 30oC.
4
The next day, the cultures obtained from E. coli
were inoculated, other 5ml of LB medium
without antibiotics and incubated for 3 hours
under the same conditions as before. Crops are
removed E. coli and A. vinelandii completed
incubation times.
helper pRK2013 Km and the recipient strain of
Azotobacter vinelandii.
During the different transformants were not
obtained enough when tested under different
conditions for the donor strain of E. coli
containing plasmid mini Tn5 Km and the
recipient strain of Azotobacter vinelandii with
and without helper plasmid, with different
ratios tested.
Example:
A. Vinelandii
is planted at
11:00 am
Incubate 24
hours at 32°C
Remove the
culture at
11:00 am
Strains of E. pUT mini Tn5
coli and seeded Helper at
8:00 pm
In subsequent trials continued testing
conditions for the donor strain of E. coli
containing plasmid mini Tn5 Sm and the
recipient strain of Azotobacter vinelandii with
and without the helper plasmid in different
proportions. It was observed that the number of
transformants was greater with the helper
plasmid such that the test was continued using
is.
Incubate for 12
hours at 37°C
Inoculate a tube with 2
° LB without antibiotic
and incubated at 37 ° C
3hr
The culture of A. vinelandii, centrifuged in two
tubes or Falcon sterile 1.5ml eppendorf tubes
and washed as often as necessary with 10 mM
MgSO4 to remove the alginate present in the
cells, (recommended to be careful when
discarding the supernatant, not to remove cells)
to obtain the tablet.
Found suitable proportions to obtain a
sufficient number of transformants using donor
strain of E. coli containing the plasmid miniTn5 Sm and the recipient strain of Azotobacter
vinelandii and the helper plasmid in ratios 1:5.1
respectively. Colonies were chopped and
replanted for conservation.
Conjugation is performed using different ratios
of the plasmid donor strain and helper receptor
in cross plates plus antibiotic selection as
appropriate.
Sample dilution:
No.
Box
1
Plates were incubated for 48 hours at 32°C,
after which time the colonies is observed with
the characteristic morphology of A. vinelandii,
and obtain a sufficient number of colonies to be
represented the genome of A. vinelandii.
Relationship
1:5:1
Proportion
25 µl de mini Tn5 Smr + 125 µl de A.
Vinelandii + 25µl pRK2013 Kmr
2
miniTn5 Smr
A. Vinelandii
pRK2013
Kmr
1
miniTn5 Smr
A. Vinelandii
pRK2013
Kmr
RESULTS
3
miniTn5 Smr
A. Vinelandii
pRK2013
Kmr
We tested different proportions between the
donor strain of E. coli containing the plasmid
mini-Tn5 Km or Sm and the recipient strain of
Azotobacter vinelandii. And moreover different
proportions between the donor strain of E. coli
containing the plasmid mini-Tn5 Km or Sm,
the strain of E. coli containing the plasmid
Conjugation plates this picture shows the
arrangement of the dilutions.
5
set for the bench of mutants of Azotobacter
vinelandii take time as it is a microorganism
that requires a culture time greater than other
microorganisms such as E . coli, and due to its
characteristics such as alginate production
make difficult the recovery of a sufficient
number of cells to perform the tests.
Several tests were required to establish the
proper proportions to obtain a sufficient
number of transconjugant colonies.
Conjugation plates after 48 hours at 32oC.
These plates were incubated for 24-48 hours at
32oC.. After obtaining the product of
conjugation is collected a certain number of
colonies on other boards. The composition of
the medium is the same as that of conjugation.
PERSPECTIVES
Enter the gus gene into the NotI site of plasmid
pUT mini Tn5 that will be used in obtaining
banking fusions.
Get banking transcriptional fusions with the
conditions established in the wild strain of
Azotobacter vinelandii.
Chop the colonies and replanting for
conservation.
CONCLUSIONS
Found suitable proportions to obtain a
sufficient number of transformants using donor
strain of E. coli containing the plasmid miniTn5 Sm and the recipient strain of Azotobacter
vinelandii and the helper plasmid in ratios 1:5.1
respectively. Were grown about 150 colonies
per plate observed the characteristic
morphology of Azotobacter vinelandii. It stung
about 1000 colonies for conservation.
The length of stay was insufficient to meet all
the objectives as the conditions and proportions
6
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