Using the Tn5 Transposon to Map Gene Locations on

pBR322 Plasmid

Abstract
Transposons are sequences of DNA that are able to move independently within the genome
with the ability to create insertion, deletion, inversion and chromosomal fusion mutations.
These features make transposons a great genetic tool for mapping segments of genomic
DNA. The main aim of our study is to isolate and map the location of the ampicillin-
resistance gene on the pBR322 plasmid using the Tn5 transposon as a mapping tool. To
carry out this experiment we firstly used the suicide phage λ::Tn5 to undergo transposon
mutagenesis with our E. Coli K12 +pBR322 cells. These insertions were then selected for on
high kanamycin plates. The plasma DNA from these cells were then isolated and
transformed into E. Coli HB101 and MC1061 strains. The transformant were then picked and
patched to isolate ampicillin sensitive and ampicillin resistant mutants. These isolates were
then digested and run on a 0.8% agarose gel to determine fragment size. From this I was
able to map two locations where the Tn5 transposon had inserted into the ampicillin gene.
For my HB101 transformed cells I mapped the location of the ampicillin gene insertion to
1951-bp along the genome. For the MC1061 strain I mapped the location of the insert to
3751-bp.

Introduction
Transposable elements are sequences of DNA that are able to move into various sites within
a genome of both prokaryotic and eukaryotic cells (Berg et al., 1983). This process between
the transposable element and the insertion site does not require extensive sequence
homology (Berg et al., 1983). These transposable elements are diverse in size and function
however, all included by two unique specific end sequences and an element-specific protein
known as transposase. Transposases allow for the movement of these elements by binding
to DNA sequences at the end of their respective elements, bringing the two ends together
through a protein oligomerization process, cutting or nicking the DNA adjacent to the end
sequences, and inserting the transposable element DNA into a DNA target site. (Berg et al.,
1983). The transposable elements along with these proteins required for transposition also
tend to carry genes encoding factors such as resistance to certain antibiotics (Berg et al.,
1983). Along with this transposable element create insertion, deletion, inversion and
chromosomal fusion mutations (Reznikoff, 2003). These features of transposons make then
a great tool for locating and mapping a gene of interest.

For our experiment we are focussed specifically on the Tn5 transposon and where it inserts
itself in the plasmid pBR322. Tn5 is a sequence of DNA approximately 5.7kb long which is
made up of two terminal inverted repeats of smaller transposition modules named IS50L
(left) and IS50R (right) which boarder three antibiotic resistance proteins for Kanamycin,
Bleomycin and Streptomycin (Reznikoff, 2003). There are also three specific restriction sites
that The IS50R sequence encodes for the transposase Tnp which can act in two opposing
ways on Tn5 (Berg et al., 1983). In cis it acts as a transposase which allows the transposition
of Tn5 whereas in trans it has the opposite affect acting instead as an inhibitor (Reznikoff,
1993). Transposition is, in general, a quite rare, highly regulated process (Reznikoff, 1993)
however, the Tn5 transposon is able to insert at high frequency's into numerous sites in
both chromosomes and plasmids (Berg et al., 1983). This mobility and ease of its antibiotic
resistance trait makes it an ideal tool for understanding the mechanisms and control of
transposition (Berg et al., 1983).

Transposons however are not autonomous replicons therefore must be delivered into the E.
Coli cell via what is known as a suicide vector. For our experiment we used the suicide
vector λ::Tn5 to mutagenize our plasmid. λ::Tn5 differ from normal phage λ as it does not
enter either the lytic (replicating and lysing the cell) or lysogenic (integrating into the
genome) cycles due to mutations. The first of these mutations are Oam and Pam which
block own vegetative replication stopping it from entering the lytic cycle. The second set of
mutations are b221 and cl which prevent it from integrating into the bacterial chromosome
thus preventing it from entering the lysogenic cycle. To grow phage’s such as λ::Tn5 it must
be done in E. Coli strains with suppressor mutations that block the mutations Oam and Pam
allowing it to propagate. In E. Coli strains that do not carry these mutations however, when
infected by λ::Tn5 the phage will not replicate however the Tn5 transposon is able to jump
to multiple targets in either the chromosome or, if it contains one, the plasmid. The phage
not being maintained allows for the selection of the rare transposon insertion by using the
antibiotic resistance marker in the case of Tn5 kanamycin resistance.

The plasmid we are attempting to map Tn5 insertion into is known as pBR322. pBR322 is a
popular cloning vector that was designed constructed for the efficient cloning and selection
off recombinant DNA molecules in E. Coli (Balbás et al., 1986). The 4361-bp DNA molecule
encodes two major genes, one is for the resistance to the antibiotic ampicillin (bla gene) and
the other resistance to antibiotic tetracycline. The plasmid also has two cleavage sites
similar to those in the Tn5 transposon. These are HindIII 29 and SaII 651.

The main aim of our study is to isolate and map the location of the ampicillin-resistance
gene on the pBR322 plasmid. We will do this by attempting to insert the Tn5 transposon
into the plasmid pBR322, specifically the ApR gene. Once we identify two isolates with this
insertion we can use them to construct a restriction map which can be used to physically
map the location of the ApR gene. We will also be mapping two non-essential regions of the
pBR322 plasmid by isolating and making two insertions outside of the Amp R gene.

Methods and Materials

All materials and methods were performed as per MICR335 lab manual.

Results

From the results of our phage titre (table 1) we can see that there was no cell lysis for E. Coli
K12 + pBR322. There is however for E. Coli strain K802 with confluent lysis occurring on the
10-5 and 10-6 plates and 19 and 5 plaques on the 10-7 and 10-8 plaques respectively. This is
expected as K12-pBR322 doesn't possess the suppressor mutations which allow the
propagation of λ::Tn5 and cannot enter the lytic cycle. K802 on the other hand does had
these mutations allow the λ::Tn5 to undergo the lytic cycle resulting in plaque formation.
The PFU calculated from the results of the K802 strain was 1.9x1010 PFU/ml. This value tells
us how many virus particles there are per ml representing the phage's biological activity.
The transposition efficiency represents the number of transposition events that occur per
phage particle. The transposition efficiency calculated given my transposon mutagenesis
results (table 2) is 9.7x10-7.

Table 1. Results from spot tittering of λ::Tn5

E. coli strain K802 E. coli strain K12 + pBR322
-5
10 Confluent Lysis No Lysis
10-6 Confluent Lysis No Lysis
-7
10 19 No Lysis
-8
10 5 No Lysis

After carrying out the transposon mutagenesis experiment we needed to determine

whether transposition of Tn5 from λ::Tn5 into E.coli/pBR322 had been successful. To do so
we plated 3 different dilutions of our mutagenized K12-pBR322 (undiluted, 1 in 10 dilution
and 1 in 100 dilution) onto two different media; one of low Kanamycin concentration (L Agar
+ Km (30 μg/ml) + Tc (15 μg/ml)) and one of high Kanamycin concentration (L Agar + Km
(200 μg/ml ) + Tc (15 μg/ml)). For the undiluted mutagenized K12-pBR322 there was an
average of 225 colonies formed across two of the plates and on the third there were too
many to count on the low kanamycin plate. On the high Kanamycin plate however, an
average of approximately 8 colonies were formed with one plate only having 4 colonies
grow. For the 1 in 10 dilution there was an average of approximately 31 colonies form on
each of the 3 low kanamycin plates and no growth on any of the high kanamycin plates. For
the 1 in 100 dilution plates there was 2 colonies form on both of the low kanamycin plates.
like the 1 in 10 dilution there was no formation of colonies on the high kanamycin plates.

L Aagr + Km (30µg/mL) + L Agar + Km (200µg/mL) +

Tc (15µg/mL) Tc (15µg/mL)

Undiluted 204, 246, TMC 10, 4, 11

1/10 Dilution 37,29,26 0,0,0
1/100 Dilution 2,2 0,0
Table 2.Plate counts of kanamycin and tetracycline resistant colonies from transposon
mutagenesis.

Following our mutagenesis experiment we pooled the colonies from both our high and low
kanamycin plates and isolated their plasmid DNA using a Zyppy Kit. We then checked the
quality of our plasmid DNA using the NanoDrop, a spectrometer that can determine the
concentration and purity of the DNA sample. The results of my low kanamycin plasmid DNA
were 41.9, 1.85 and 1.52 which is around what we expect to see for good quality DNA. The
results of my high kanamycin plasmid DNA however were 1.1, 0.89 and 0.35. These results
were well below what we expected to see indicating poor quality DNA. To further test the
quality, we ran both mine and my partners plasmid DNA along a 0.8% agarose gel (figure 1).
The bright band that has run the furthest along the gel is the supercoiled plasmid. Going up
the gel the second band is linear plasmid DNA as sometimes the plasmid DNA can be cut.
The little lines at the very top of the gel represent plasmid DNA that has been nicked. In
columns 2,3 and 5, which includes both my partners high and low and my low, we can see
that there is a good amount of DNA. In the last column which is my high DNA we don’t see
that given the less bright bands. This just backs up our findings from the NanoDrop which
also suggested my high plasmid DNA was of poor quality/concentration. Because of this I
decided to use my partners plasmid DNA from the columns 2 and 3 for the transformation.

Figure 1. Agarose gel electrophoresis (0.8% agarose) of uncut plasmid DNA run for 1 – 2
hours. Lane 1 represents DNA standard. Lane 3 is low pool plasmid DNA. Lane 4 is high pool
plasmid DNA. Lane 5 is low pool plasmid DNA. Lane 6 is high pool plasmid DNA.

We then transformed our isolated plasmid DNA into either E. Coli strain HB101 or MC1061.
For my experiment I transformed into strain HB101. After transformation we spread our
transformed cells onto 5 separate media of L agar + Km (3015 μg/ml) + Tc (15 μg/ml)). These
plates would select for transformants that had taken up the plasmid with the Tn5
transposon insertion. We also plated the cells on to one plate each of L agar + Tc (15 μg/ml)
that would act as a positive control seeing if the transformation had occurred and pBR322
plasmids had been taken up by the E. Coli regardless of if they had the Tn5 transposon. We
then plated 1/10 dilution and 1/100 dilutions for each set of transformed cells onto 1 plate
of each media. We also plated a DNA sterility control on one of each media (Table 3). For
the High Kanamycin undiluted cells, a total of 3 colonies formed across all 5 of the Km + Tc
plates. For the Tc only, plate however there were to many colonies to count on the one
plate. For the low undiluted, high 1/10 dilution, low 1/10, high 1/100 and low 1/100 cells
not colonies formed on any of the Km + Tc media. On the Tc only, media however there
were TMTC, 92, 72, 4 and 5 colonies of each of the respective plates. These results tell me
that although transformation had been successful, given the growth on the Tc only plates,
there were very limited transformants had the Tn5 insertion in the plasmid. This tells me
that my transposon mutagenesis experiment did not work well.
Table 3. Transformants of strain HB101
L-agar + Tc + Km L-agar + Tc
High (Undiluted) 0,0,1,2,0 TMTC
Low (Undiluted) 0,0,0,0,0 TMTC
High (1/10) 0 92
Low (1/10) 0 72
High (1/100) 0 4
Low (1/100) 0 5
No DNA control 0 0
Sterility Control 0 3

Because there wasn’t enough colonies containing the pBR322::Tn5 plasmids grown on my
Km + Tc plates, and I didn’t have time to repeat, I instead used colonies provided to me by
the demonstrators. To screen these transformants and determine whether or not the Tn5
had inserted into the AmpR (bla) gene or not we carried out a pick and patch. This works by
picking a transformant with a tooth pick and patching it onto firstly L agar + Km + Tc plates
and then onto an L agar + Km + Ap plate. The L agar + Km + Tc plates would select for all
plasmids with the Tn5 insertion and the L agar + Km + Ap plate would only select for
transformants that didn’t have the Tn5 in the Ap gene. By comparing the two we could
determine if any were ApS as they wouldn’t grow on the L agar + Km + Ap plate. I firstly pick
and patched 70 different transformants onto either media. The results for this initial test
showed I had no AmpS mutants, a frequency of insertion into the bla gene of 0%. Because of
this I pick and patched another 100 different colonies but once again found no Amp s
mutants only AmpR. To carry on with the experiment an ApSKmRTcR colony from transformed
HB101 was provided by a demonstrator. I then single colony purified my Ap SKmRTcR and
ApRKmRTcR and my partners ApSKmRTcR and ApRKmRTcR from the transformed MC1061 strain.

Table 4. Pick and Patch results, ampicillin sensitive and resistant colonies.
Number of Number of ApR Number of ApS Frequency of
colonies Isolates Isolates Insertion (%)
patched
Attempt 1 70 70 70 0
Attempt 2 100 100 100 0

To begin identifying the site of insertion of the Tn5 I firstly extracted the plasmid DNA using
an QIAPrep minikit. Once extracted I restriction endonuclease digested both plasmids from
the MC1061 and HB101. For each of these strains I did a Hindlll single digest and a Hind/Sall
double digest. The digested DNA along with DNA standard was then run on a 0.8% agarose
electrophoresis gel for 1-2 hours. The DNA standard which had known fragment sizes was
then used to create a standard curve by plotting by plotting the distance travelled (mm) of
each fragment from the wells against the fragment size. This standard curve was then used
to estimate the sizes of the fragments produced by the digest. We already knew that the
plasmids digested by the single Hinlll restriction enzyme would always include a fragment
3.4kb long as this is the distance between the two Hinlll restriction site on the Tn5
transposon. For the double Hinlll/Sall digest there will be three known fragments produced.
These are a 1.5kb fragments which is the distance between one of the Hinlll sites on the Tn5
transposon and the Sall site on Tn5. The next is a 1.9kb fragment which is the distance
between the other Hinlll site and the Sall site on Tn5. The 0.6kb fragment is the distance
between the Hinlll and Sall sites on the pBR322 plasmid. For the remaining bands the size is
estimated by measuring the distance they travelled (mm) from the wells against the
standard curve produced by the DNA standard. We digest the plasmid DNA from
transformed HB101 (figure 3) and transformed MC1061 (figure 4) separately.

Figure 2. Agarose gel electrophoresis (0.8% agarose) of Tn5 mutagenized pBR322:Tn5

plasmid DNA from transformed HB101 digested with HindIII and SaII restriction enzymes.
The gel was run for 1-2 hours. In lane one is DNA standard with known fragment sizes. This
is used to create a standard curve which is used to predict the sizes of the other unknown
fragments. In lane two is a single digest of Aps HB101 with the HindIII restriction enzyme.
Lane three is a double digest of Aps HB101 with the HindIII/Sall restriction enzyme. lane four
is a single digest of ApR HB101. Lane five is a double digest of ApR HB101.
Figure 3. Agarose gel electrophoresis (0.8% agarose) of Tn5 mutagenized pBR322:Tn5
plasmid DNA from transformed MC1061 digested with HindIII and SaII restriction enzymes.
The gel was run for 1-2 hours. In lane one is DNA standard with known fragment sizes. This
is used to create a standard curve which is used to predict the sizes of the other unknown
fragments. In lane two is a single digest of Aps MC1061with the HindIII restriction enzyme.
Lane three is a double digest of Aps MC1061 with the HindIII/Sall restriction enzyme. lane
four is a single digest of ApR MC1061. Lane five is a double digest of ApR MC1061. Lane 6 is a
single digest of the unmutated pBR322 plasmid. Lane 7 is a double digest of the unmutated
pBR322 plasmid.
Fr
ag
me
nt
Siz
e
(b
p)

Distance Travelled (mm)

Figure 4. Standard curve of DNA Standard of fragment size (bp) vs distance travelled (mm)
from gel 1 (figure 2.)
Fr
ag
me
nt
Siz
e
(b
p)

Distance travelled (mm)

Figure 5. Standard curve of DNA Standard of fragment size (bp) vs distance travelled (mm)
from gel 2 (figure 3.)

Table 5. Mapping results for HB101 Aps based on distance travelled on gel (figure 1) and
DNA standard curve (figure4).
Fragment Estimated from Standard Curve Adjusted to 10.2kb
Number
Hindlll Hindlll/Sall Hindlll Hindlll/Sall
1 3.7kb 3.5kb 3.7kb 3.7kb
2 3.5kb 2.5kb 3.4kb 2.5kb
3 3.1kb 1.9kb 3.1kb 1.9kb
4 1.4kb 1.5kb
5 0.5kb 0.6kb

Table 6. Mapping results for HB101 ApR based on distance travelled on gel (figure 1) and
DNA standard curve (figure4).
Fragment Estimated from Standard Curve Adjusted to 10.2kb
Number
Hindlll Hindlll/Sall Hindlll Hindlll/Sall
1 4.9kb 4.2kb 4.9kb 4.3kb
2 3.4kb 2.0kb 3.4kb 1.9kb
3 1.9kb 1.9kb 1.9kb 1.9kb
 4 1.5kb 1.5kb
5 0.8kb 0.6kb

Table 7. Mapping results for MC1061 Aps based on distance travelled on gel (figure 2) and
DNA standard curve (figure 5).
Fragment Estimated from Standard Curve Adjusted to 10.2kb
Number
Hindlll Hindlll/Sall Hindlll Hindlll/Sall
1 4.7kb 4.2kb 4.9kb 4.3kb
2 3.4kb 1.9kb 3.4kb 1.9kb
3 1.9kb 1.9kb 1.9kb 1.9kb
4 1.5kb 1.5kb
5 0.5kb 0.6kb

Table 8. Mapping results for MC1061 ApR based on distance travelled on gel (figure 2) and
DNA standard curve (figure 5).
Fragment Estimated from Standard Curve Adjusted to 10.2kb
Number
Hindlll Hindlll/Sall Hindlll Hindlll/Sall
1 3.7kb 4.2kb 3.7kb 3.7kb
2 3.5kb 2.7kb 3.4kb 2.5kb
3 3.1kb 1.9kb 3.1kb 1.9kb
4 1.5kb 1.5kb
5 0.6kb 0.6kb

Figure 6. Map locations of fragment sizes for HB101 Aps based on figures from table 5.
Figure 7. Map locations of fragment sizes for HB101 Ap R based on figures from table 6.

Figure 8. Map locations of fragment sizes for MC1061 Ap R based on figures from table 8.

Figure 9. Map locations of fragment sizes for MC101 Ap S based on figures from table 7.

Looking at our mapping results we can see we mapped our two non-essential Tn5 insertions
at the locations 3751 (figure 7) and 1951 (figure 8) along the genome. The two ampicillin
sensitive isolates had their insertions mapped at 1951 (figure 6) for the HB101 strain and
3751 (figure 9) for the MC1061 strain. This tells is that the ampicillin resistance (bla) genes
are at these locations for each respective strain. These results however are a rough estimate
based on our best guesses from a standard curve line. Therefore, the results may not be
that accurate.

Discussion
The pBR322 plasmid is 4361 Bp long whereas the chromosome is 4.4mb long . This means
there is a far higher chance of the Tn5 inserting into the chromosome as it has more targets.
This is why in the results of the transposon mutagenesis there were a lower number of
colonies with the Tn5 insertion in the plasmid which is what we are looking for. This is why
we plated the cells from the transposon mutagenesis on the High and Low kanamycin
plates. The high kanamycin(200ug) plates selected more for the cells that had the Tn5
inserted into pBR322 rather than the chromosome. The reason for this is that the pBR322
plasmid has higher copy number than the chromosome meaning it is able to replicate more
in the cell. The plasmid copy number of pBR322 in E. coli cells are 15-20 copies per cell
which mean theoretically (Balbás 2004). if Tn5 is mutagenized into the plasmid, there will be
15-20X the amount of kanamycin resistance. Therefore, at on the high kanamycin plates the
cells with the insertions in the plasmid will have more resistance to the kanamycin and will
be more likely to survive than the cells with insertions in the chromosome. This is in line
with the literature. In a study by Sasakaw et al. (1982) they found that cells harbouring Tn5
in the multicopy pBR322 are resistant to higher levels of neomycin (another antibiotic
resistance gene found in Tn5) than isogenic cells containing Tn5 in a single chromosomal
locus (Sasakaw et al. 1982). Hence why in our results we saw a larger number of
mutagenized colonies growing on the low kanamycin plates in comparison to the high
levels. This does not mean however that all colonies on the high kanamycin plate will have
insertions in the pBR322 plasmid but there is a far higher likelihood.

The efficiency of transposition tells us the number of transposition events that will occur per
phage. The value calculated for my transposon mutagenesis was 9.7x10-7. This value is in
line with the literature as the average efficiency of transposition for prokaryotic transposons
such as Tn5 is 1x107 transposition events per phage particle (Kleckner, 1981).

For our experiment we used two different E. coli strains for the transformation step. These
are HB101 and MC1061. Because I only used the HB101 strain for my experiment to
compare the two we have to look at the class results. From these results we can see for
transformation, those using the HB101 strain on average had less successes full
transformation attempts and a lower number of transformants on their plates. RecA is a
regulator of recombination in E. Coli cells (Hanahan et al., 1991). HB101 strain has a
mutation in the recA gene making it recA-. This mutation dispenses stability to DNA
sequences and prevents recombination of the plasmid into the chromosome (Hanahan et
al., 1991). This prevention of recombination suggests it should be more efficient for
transformation. Looking at the class data we see this is quite the opposite with the MC1061
strain being more efficient and obtaining more transformants. MC1061 unlike HB101
doesn’t have a mutation in the recA gene (recA+). A mutation in the recA gene however
compromises the DNA repair process. A compromised DNA repair process is typical of
recombination deficient strains which may explain why the transformation was less efficient
in the HB101 strain (Hanahan, 1983). Unlike the HB101 strain the MC1061 strain has a galU
mutation. The galU gene produces a protein called UTP which is involved in the
synthetisation of lipopolysaccharide molecules (LPS) on the outer membrane. The changing
structure and function of the LPS molecules increase the exposure of DNA binding sites
which are thought to make them better recipients of plasmid DNA (Mclachlan 1984). Hence
this could be the reason why MC1061 is more efficient for transformation than HB101.

From my own pick and patch results we saw 85 AmpR and 0 AmpS isolates, a frequency of
insertion into the insertion of 0% into the bla (AmpR) gene. As the true insertion frequency is
not 0% to get an idea of the true insertion frequency of the Tn5 into the AmpR gene for the
experiment I compared the class results. From the class pick and patch results the average
frequency of insertion was 7.2%. Looking at the sizes of the Ap gene and pBR322 plasmid
however, this is not what we would expect to see. The Ap gene is 861-bp in size which
compared to the 4361-bp total size of the pBR322 plasmid makes up approximately 20% of
it. If Tn5 did insert randomly into the plasmid we would therefore expect to see an insertion
frequency of close to 20%. Looking through the literature confirms that Tn5 insertion is not
quite random. In the paper Berg et al., (1983) when doing the same transposon mutagenesis
experiment they found that Tn5 tended to insert into the tet gene of pBR322. Of 75
independent insertions 21 were found in this specific site on the tet gene. Along with this 26
were distributed along 4 different hotspots. None of these other hotspots however are
found in the amp gene (Berg et al., 1983. The remaining 28 were found in 23 different sites.
When comparing these results to the class monomer insertion map however we see there
are only 2 insertions at the main tet hotspot. This is at a far lower frequency than found in
the Berg study. The reason for this may be due to tetracycline being used to select for cells
containing the plasmid in both the transformation and mutagenesis. Therefore, since we are
selecting for tetr we are in turn selecting against plasmids with Tn5 insertions in this hotspot.
Another feature of these hotspots is they are all 9-bp segments that are bordered by GC
base pairs. This suggests there is a GC cutting preference during Tn5 transposition (Berg et
al., 1983).

A dimer is formed when two monomers join together creating a single larger structure. In
this case a dimer of pBR322 can be formed from two monomeric pBR322 plasmids. The
reason these dimers are able to occur with pBR322 is due to the plasmid lacking the cis
acting cer sequence which is required to resolve plasmid multimers. The way these are
usually resolved is through site specific recombination (Balbás & Bolívar, 2004). Plasmids in
dimer form also causes instability as well as the dimer cells grow slower to their monomer
counterparts ((Mazin, 1996). . For or mutagenized pBR322 a normal monomer (Tn5 +
pBR322) is 10179bp in size however a dimer is 14540bp. This can be identified through gel
electrophoresis. If it dimer is singly digested it will have 4 bands as opposed to the 3 for a
monomer. If it is double digested there will be 7 bands as opposed to 5. From our class
results we can see that 18 individuals had Tn5 insert into dimeric pBR322. This is far less
than those that had insertions into dimeric DNA. As mentioned the instability of the plasmid
and the slower growth rate may be why we see this.
To conclude, using the Tn5 transposon we were successfully able to isolate and map the
location of the ampicillin-resistance (bla) gene on the pBR322 plasmid along with other non-
essential genes. Doing this showed just how useful a transposon, such as Tn5, can be to map
gene within a genome given both its mobility and the antibiotic resistance. If I were to do
this experiment again I would base my experiment more on that in the paper Blair,. 1983
and focus more on the insertion of the Tn5 transposon in the tet gene.

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