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1 DNA Amplification, Cloning, and Sequencing

1 DNA Amplification, Cloning, and Sequencing

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Genetic Strategies on Kennedy Pathway to Improve Triacylglycerol Production. . .

127

Table 4
Strains and plasmids described in this chapter
Strain or plasmid

Description

Source

E. coli K-12 F_ lacU169 (ϕ80lacZΔM15) endA1
recA1 hsdR17 deoR supE44 thi-1-l2 gyrA96
relA1. Use for cloning and subcloning
purposes
endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+
Δ(lac-proAB) e14- [F’ traD36 proAB+ lacIq
lacZΔM15] hsdR17(rK-mK+). Use for cloning
purposes

[56]

Strains
E. coli
DH5α

JM109

Promega

Rhodococcus
R. jostii
RHA1
RHA1 pJAM2
RHA1 pJAM2/RO00075
RHA1 pTip-QC2
RHA1 pTip-QC2/RO00075

Parental strain
RHA1 derivative carrying pJAM2 plasmid, used
as control strain; KmR
RHA1 derivative carrying pJAM2/ro00075; KmR
RHA1 derivative carrying pTip-QC2 plasmid,
used as control strain; CmR
RHA1 derivative carrying pTip-QC2 /ro00075;
CmR, ThioR

[57]
[12]

Parental strain
PD630 derivative carrying pJAM2 plasmid, used
as control strain; KmR
PD630 derivative carrying pJAM2/ro00075;
KmR
PD630 derivative carrying pTip-QC2 plasmid,
used as control strain; CmR
PD630 derivative carrying pTip-QC2 /ro00075;
CmR, ThioR

DSM 44193
[11]

PD630 derivative carrying pJAM2/atf1; KmR
PD630 derivative carrying pPR27ace/atf2; GmR
PD630 derivative carrying integrative plasmid
pMVace/atf2; KmR

[11]
[11]
[11]

Linear plasmid used for cloning PCR products;
ApR
Shuttle E. coli–Mycobacterium–Rhodococcus with
Pace promoter; KmR
pJAM2 derivatives carrying ro00075 gene under
control of Pace ; KmR
Expression vector for Rhodococcus with PtipA
promoter, repAB (pRE2895); CmR

Promega

[12]
[12]
[12]

R. opacus
PD630
PD630 pJAM2
PD630 pJAM2/RO00075
PD630 pTip-QC2
PD630 pTipQC2/RO00075
PD630 pJAM2/atf1
PD630 pPR27ace/atf2
PD630 pMVace/atf2

[12]
[12]
[12]

Plasmids
pGEM-T-easy vector
pJAM2
pJAM2/ro00075
pTip-QC2

[32]
[12]
[36]
(continued)

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Martı´n A. Herna´ndez and He´ctor M. Alvarez

Table 4
(continued)
Strain or plasmid

Description

Source

pTip-QC2/ro00075

pTip-QC2 carrying ro0075 gene under control of
PtipA; CmR
pJAM2 derivatives carrying atf1 gene under
control of Pace ; KmR
E. coli–Mycobacterium shuttle vector, oriM
temps, sacB, XylE; GmR
pPR27 derivatives carrying atf2 gene under
control of Pace ; GmR
pTip-QC2 carrying atf2 gene under control of
PtipA; CmR
Integrative plasmid derived from pMV306 and
containing a Pace promoter; KmR
pMVace carrying atf2 gene under control of Pace;
KmR

[12]

pJAM2/atf1
pPR27
pPR27ace/atf2
pTip-QC2/atf2
pMVace (pMR22)
pMVace/atf2

[11]
[39]
[11]
[11]
[45], this chapter
[11]

of DNA are prepared by a commercial mini-prep extraction and
stored at À20 C.
2. Restriction endonucleases and other enzymes: BamHI, XbaI,
HindIII, NdeI, NotI, T4 DNA ligase, and its buffers.
All enzymes may be purchased from any supplier.
3. Other reagents: Agarose molecular grade and DNA purification kit (from enzymatic reactions and agarose gels).
2.3 Electroporation
of Rhodococcus Cells

1. Electroporator: Electroporation of Rhodococcus cells requires a
high-voltage electroporator. Good efficiencies have been
obtained with the model 2510 electroporator (EppendorfNetheler-Hinz, Hamburg, Germany).
2. Cuvettes of 400 μL with electrode gaps of 2 mm.
3. Cold bidistilled water and ice containers.
4. LB media and LB media supplemented with glycine (0.85% w/v)
and sucrose (1% w/v).

2.4 Bacterial
Cultures

1. For routine growth of E. coli and Rhodococcus strains, use solid
or liquid Luria-Broth (LB) media and incubate them at 37 C
and 28 C, respectively. LB medium may be purchased for any
supplier or prepared from individual components as:
(a) 10 g/L peptone, 5 g/L yeast extract, and 5 g/L NaCl in
distilled water

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129

2. To allow TAG accumulation, use a minimal salt medium
(MSM) prepared from individual components as:
(a) 9 g/L Na2HPO4. 12 H2O, 1.5 g/L KH2PO4, 0.1 g/L
NH4Cl, 0.2 g/L MgSO4. 7 H2O, 20 mg/L CaCl2.
2 H2O, 1.2 mg/L Fe(III)NH4-citrate, and 0.1 mL/L of
trace element solution SL6 (10 mg/L ZnSO4. 7 H2O,
3 mg/L MnCl2. 4 H2O, 30 mg/L H3BO3, 20 mg/L
CoCl2. 6 H2O, 1 mg/L CuCl2. 2 H2O, 2 mg/L NiCl2.
6 H2O, 3 mg/L Na2MoO4. 2 H2O) in distilled water. Use
glucose as sole carbon source at a final concentration of 1%
(w/v) in MSM media (see Note 2). Use 0.1 g/L of ammonium chloride in MSM0.1 culture media. Do not add
ammonium chloride for preparing MSM0 culture media
(see Note 3).
3. Use agar-agar for solid media at a final concentration of 1.4%
(w/v). Autoclave under standard condition and adjust the pH
to 7. If necessary, add antibiotics after autoclaving at different
final concentrations as follows: 100 μg/mL ampicillin (Ap),
50 μg/mL kanamycin (Km), 5 μg/mL gentamicin (Gm), and
34 μg/mL chloramphenicol (Cm) for both E. coli and Rhodococcus cell cultures.
4. For overexpression analysis of genes under the acetamidase
promoter Pace of pJAM2/pPR27ace/pMVace vectors, add
0.5% (w/v) of acetamide to cell cultures at time zero. For
overexpression analysis of genes under the thiostrepton promoter PTipA of pTip-QC2 vector, add 1μg/mL of thiostrepton
to cell cultures at time zero (see Note 4).
2.5

Lipid Analysis

1. Solvents for lipid extraction: Chloroform and methanol. HPLC
grade.
2. Solvents for TLC development: Hexane, diethyl ether, and
acetic acid. HPLC grade.
3. TLC plates: Silica gel 60 F254 plates (Merck). Activate at 65 C
before use for 30 min.
4. Pure iodine crystals (see Note 5).
5. Reference lipids: Use tripalmitin dissolved in chloroform as
TAG standard at a final concentration of 1 mg/mL.

3

Methods
This section describes a protocol for TAG overproduction in
oleaginous Rhodococcus strains by overexpression of three genes:
ro00075, atf1, and atf2. For this, construction of several recombinant molecules using the vectors pJAM2, pTip-QC2, pPR27ace,
and pMVace is described. Some of these constructions contain a

130

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C-terminal or N-terminal 6ÂHis-tag fusion, and gene products
can be detected by Western blotting. The resulted recombinant
strains are then subjected to semiquantitative TLC analysis of
TAG content.
3.1 DNA
Amplification, Cloning,
and Sequencing

The first step for construction of recombinant molecules is the
correct amplification of complete coding sequence of interest. For
specific DNA amplification of ro00075, atf1, and atf2 genes
described in this chapter, perform PCR assays with specific primers
listed in Table 4. Use total DNA of R. jostii RHA1 and R. opacus
PD630 as template for amplification of ro00075 and the two atf
genes, respectively. In all cases, use the following thermocycler
parameters: 5 min at 94 C, 30 cycles of 1 min at 94 C, 30 s at
60 C, 1 min at 72 C, and finally 5 min at 72 C. After checking on
1% agarose gel, clone the PCR products into any TA cloning vector,
transform in E. coli, and perform screening of colonies by the blue/
white color selection and then by colony PCR. Select positive
clones and verify the correct amplification by DNA sequencing of
purified plasmid.

3.2 Subcloning
Strategies

To achieve overexpression of ro00075 in R. jostii RHA1 and
R. opacus PD630 strains under an inducible acetamide promoter
Pace (also called Pami), proceed as follows:

3.2.1 Subcloning of
ro00075 Gene in pJAM2
Vector (see Fig. 2)

1. Cut both purified plasmids pJAM2 and pGEM-T-easy/
ro00075 with the enzymes BamHI and XbaI. Purify the linearized pJAM2 vector directly from the enzymatic reaction. After
running the cut pGEM-T-easy/ro00075 on a 1% agarose gel,
purify the ro00075 gene of ~700 bp from TBE agarose gel
using a DNA purification kit.
2. To a 0.5-mL tube, add the cut pJAM2 vector and the ro00075
insert at molar ratios ranging from 1:1 to 1:2, 10Â ligation
buffer, and dH20 to 10 μL. Add T4 DNA ligase, mix, and
incubate overnight at 8 C.
3. Transform in E. coli DH5α and check kanamycin-resistant
colonies by colony PCR using the primers aceF/RO00075R
(Table 4).

3.2.2 Subcloning of
ro00075 Gene in pTip-QC2
Vector (see Fig. 2)

To achieve overexpression of ro00075 in R. jostii RHA1 and R.
opacus PD630 strains under an inducible thiostrepton promoter
(PTipA), proceed as follows:
1. Cut both plasmids pTip-QC2 vector and pJAM2/ro00075
obtained above with the enzymes BamHI and HindIII (see
Note 6). Purify the linearized pTip-QC2 vector directly from
the enzymatic reaction using a DNA purification kit. After
running the cut pJAM2/ro00075 on a 0.8% agarose gel, purify
the fragment of ~2.27 kb carrying the ro00075 gene from the
gel using a DNA purification kit.

Genetic Strategies on Kennedy Pathway to Improve Triacylglycerol Production. . .

131

Fig. 2 Overview of molecular strategies described in this chapter for ro00075 and atf1 overexpression in
pJAM2 and pTip-QC2 vectors. The transferred molecules to Rhodococcus cells are marked with an asterisk.
The size of plasmids is not to scale

2. To a 0.5-mL tube, add the cut pTip-QC2 vector and the
ro00075-carrying fragment at molar ratios ranging from 1:1
to 1:2, 10Â ligation buffer, and dH20 to 10 μL. Add T4 DNA
ligase, mix, and incubate overnight at 8 C.
3. Transform in E. coli DH5α and check ampicillin-resistant colonies by colony PCR using the primers thioF/RO00075R
(Table 4).
3.2.3 Subcloning of atf1
Gene in pJAM2 Vector (see
Fig. 2)

To achieve overexpression of atf1 in Rhodococcus opacus PD630
strain under an inducible acetamide promoter Pace, proceed as
follows:
1. Cut both purified plasmids pJAM2 and pGEM-T-easy/atf1
with the enzymes BamHI and XbaI. Purify the linearized

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Martı´n A. Herna´ndez and He´ctor M. Alvarez

pJAM2 vector directly from the enzymatic reaction. After running the cut pGEM-T-easy/atf1 on a 1% agarose gel, purify the
atf1 gene of ~1,460 bp from TBE agarose gel using a DNA
purification kit.
2. To a 0.5-mL tube, add the cut pJAM2 vector and the atf1
insert at molar ratios ranging from 1:1 to 1:2, 10Â ligation
buffer, and dH20 to 10 μL. Add T4 DNA ligase, mix, and
incubate overnight at 8 C.
3. Transform in E. coli DH5α and check kanamycin-resistant
colonies by colony PCR using the primers aceF/atf1MHR
(Table 4).
3.2.4 Subcloning of atf2
Gene in the Integrative
Plasmid pMVace Vector (see
Fig. 3)

To achieve overexpression of atf2 under an inducible acetamide
promoter Pace as a single extra copy in Rhodococcus opacus PD630
strain by using an integrative plasmid, proceed as follows:
1. Cut both purified plasmids pMVace and pGEM-T-easy/atf2
with the enzymes NdeI and HindIII. Purify the linearized
pMVace vector directly from the enzymatic reaction. After running the cut pGEM-T-easy/atf2 on a 1% agarose gel, purify the
atf2 gene of ~1,380 bp from TBE agarose gel using a DNA
purification kit.
2. To a 0.5-mL tube, add the cut pMVace vector and the atf2
insert at molar ratios ranging from 1:1 to 1:2, 10Â ligation
buffer, and dH20 to 10 μL. Add T4 DNA ligase, mix, and
incubate overnight at 8 C.
3. Transform in E. coli DH5α and check kanamycin-resistant
colonies by colony PCR using the primers aceF/atf2MHR
(Table 4).

3.2.5 Subcloning of atf2
Gene in pPR27ace Vector
(see Fig. 3)

To achieve overexpression of atf2 in Rhodococcus opacus PD630
strain under an inducible acetamide promoter Pace in a replicative
plasmid version with a gentamicin resistance (see Note 7), proceed
as follows:
1. Cut both purified plasmids pPR27 and pMVace/atf2 obtained
above with the enzymes BamHI and NotI. Purify the linearized
pPR27 vector directly from the enzymatic reaction using a
DNA purification kit. After running the cut pMVace/atf2 in a
0.8% agarose gel, purify the fragment of ~3.6 kb carrying the
atf2 gene under Pace promoter from the agarose gel using a
DNA purification kit (see Note 8).
2. To a 0.5-mL tube, add the cut pPR27 vector and the atf2carrying fragment at molar ratios ranging from 1:1 to 1:2, 10Â
ligation buffer, and dH20 to 10 μL. Add T4 DNA ligase, mix,
and incubate overnight at 8 C.

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133

Fig. 3 Overview of molecular strategies described in this chapter for atf2 overexpression in pMVace, pPR27ace,
and pTip-QC2 vectors. The transferred molecules to Rhodococcus cells are marked with an asterisk. The size
of plasmids is not to scale

3. Transform in E. coli DH5α and check gentamicin-resistant
colonies by colony PCR using the primers aceF/atf2MHR
(Table 4).
3.2.6 Subcloning of atf2
Gene in pTip-QC2 Vector
(See Fig. 3)

To achieve overexpression of atf2 in Rhodococcus opacus PD630
strains under an inducible thiostrepton promoter (PTipA), proceed
as follows:
1. Cut both purified plasmids pTip-QC2 and pMVace/atf2
obtained above with the enzymes NdeI and HindIII (see
Note 9). Purify the linearized pTip-QC2 vector directly from
the enzymatic reaction using a DNA purification kit. After
running the cut pMVace/atf2 in a 0.8% agarose gel, purify the
atf2 fragment of ~1,364 bp from the agarose gel using a DNA
purification kit.
2. To a 0.5-mL tube, add the cut pTip-QC2 vector and the atf2
insert at molar ratios ranging from 1:1 to 1:2, 10Â ligation
buffer, and dH20 to 10 μL. Add T4 DNA ligase, mix, and
incubate overnight at 8 C.
3. Transform in E. coli DH5α and check ampicillin-resistant colonies
by colony PCR using the primers thioF/atf2MHR (Table 4).

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Martı´n A. Herna´ndez and He´ctor M. Alvarez

3.3 Electroporation
of Rhodococcus Cells

Transfer all recombinant plasmids to R. jostii RHA1 and R. opacus
PD630 by electroporation based on the method described by
Kalscheuer et al. (1999) [17]:
1. To obtain electrocompetent Rhodococcus cells, inoculate 25 mL
of LB medium in a 100 mL Erlenmeyer flask supplemented
with 0.85% (w/v) glycine and 1% (w/v) sucrose with 1 mL of
an overnight LB preculture and grown at 28 C to an optical
density of 0.5 at 600 nm.
2. Harvest cells, wash twice with ice-cold bidistilled H2O, and
concentrate 20-fold in ice-cold bidistilled H2O.
3. Immediately before the electroporation, mix 400 μL of competent cells with DNA (0.1–1 μg/mL) and preincubate at
40 C for 5 min. Perform electroporation in cuvettes with
gaps of 2 mm and the following settings: 10 kV/cm, 600 Ω,
and 25 μF. Accept time constants of 3–5 ms only. Dilute
immediately pulsed cells with 600 mL of LB and incubate at
28 C for 4 h (see Note 10) before plating on appropriate
selective media. Check plates after 3–4 days.
Additionally, transfer to R. jostii RHA1 and R. opacus PD630
the correspondent empty plasmids to obtain control cells. Perform
colony PCR of at least five individual clones to check the presence
of each gene in the recombinant plasmids. For this, use the set
primers aceF/RO00075R, aceF/atf1MHR, and aceF/atf1MHR
to check ro00075, atf1, and atf2 genes under Pace and thioF/
RO00075R, thioF/atf1MHR, and thioF/atf2MHR to check
ro00075, atf1, and atf2 genes under PTipA promoters, respectively
(see Table 4). Select one positive clone of each recombinant strain
for further analysis.

3.4 Bacterial
Cultures

After obtaining recombinant strains, the next step is to analyze the
phenotype. The next protocol is focused particularly in the cellular
conditions for TAG accumulation. For this, proceed as follows:
1. Pick isolated colonies of both recombinant Rhodococcus strains
and its respective controls (cells containing the empty vector)
from fresh plates and transfer into 20 mL liquid Luria-Broth
(LB) medium and cultivate cells aerobically at 28 C and
200 rpm overnight. Use the appropriate antibiotic (see Sect. 2).
2. Inoculate fresh LB media with 2 mL of the overnight precultures and incubate again at 28 C and 200 rpm for 24 h.
Harvest cells and wash twice with sterile physiological solution
(NaCl 0.85% w/v).
3. Inoculate 1 mL of resuspended cells (DO600 4) into 20 mL of
MSM0.1 media or resuspend all harvest cells in 25 mL MSM0.
Use glucose as sole carbon source at a final concentration of
1% (w/v).

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135

4. Add the appropriate antibiotic and inducers as described in
Materials section.
5. Incubate cell cultures at 28 C and 200 rpm for 48 h. Harvest
cells and lyophilize or dry to constant weight at 37 C
(see Note 11).
3.5 TLC Lipid
Analysis

To semiquantitatively analyze the total intracellular lipids in Rhodococcus cells, perform the next steps as:
1. Weigh 5 mg of dried cells (see Note 12) in 1.5 mL tubes and
extract lipids with 300 μL of mix chloroform/methanol (2:1,
v/v) for 90–120 min. Vortex vigorously each for 30 min.
2. Centrifuge the extracts at 13,000 rpm for 5 min. Remove
100 μL of chloroformic phase to fresh tubes. Perform TLC
analysis by loading 20 μL of chloroformic phase on silica gel 60
F254 plates (Merck).
3. Run TLC with hexane/diethyl ether/acetic acid (80:20:1, v/v/v)
as mobile phase. Compare spots with a reference substance of TAG
(see Sect. 2).
4. Visualize different lipid fractions using iodine vapor (see an
example in Fig. 4).

Fig. 4 Example of a TLC analysis of TAG from whole-cell extracts overexpressing
ro00075 and atf2 in pTip-QC2 vector. (I and III) PD630 cells with empty pTipQC2, (II) PD630 cells carrying pTip-QC2/ro00075, and (IV) PD630 cells carrying
pTip-QC2/atf2

136

Martı´n A. Herna´ndez and He´ctor M. Alvarez

Additionally, TAG content can be quantified as total fatty acids
from dried Rhodococcus cells by GC analysis or a colorimetric
method as described in previous works [58, 59].

4

Notes
1. Alternative restriction sites can be used in primers for subcloning purposes according to the cloning sites in vector systems
described in this chapter [32, 36, 39].
2. In this chapter, glucose is proposed as carbon source; however,
another carbon source can be used at appropriate final concentration [2, 3].
3. MSM0 medium is used to analyze more accurately TAG biosynthesis, independently of the cellular growth. Under these
conditions, cell growth is impaired because the nitrogen source
is lacking in the medium; thus, cells utilize the available carbon
source principally for TAG biosynthesis and accumulation. We
observed that the use of MSM0 medium containing an excess
of a carbon source promotes more pronounced differences in
TAG concentrations between control and recombinant cells.
4. To our experience, we observed a better recombinant protein
production when the inducer is added at the beginning of the
cell cultivation without a loss of cellular viability; however, variations in the addition times and final concentrations of inducers
can be applied for different genes in the same expression vectors.
5. Because oleaginous Rhodococcus cells are able to accumulate
large amounts of neutral lipids (mainly TAG and minor
amounts of free fatty acids, DAG and MAG), iodine vapors
are sufficient for a rapid and sensitive visualization of them.
In addition, the sensibility can be increased putting preiodine-stained plates under UV light of a transilluminator.
Other techniques can be used to visualize lipids on TLC plates
as the cupric-phosphoric staining.
6. Please note that ro00075 gene (~700 bp) can be also directly
amplified and subcloned into pTip-QC2 vector using the
appropriate restriction enzymes such as NdeI/HindIII and
BamHI/HindIII, among others.
7. Please note that atf2 gene (~1,380 bp) can be directly amplified
and subcloned into pJAM2 vector (which is also a replicative
plasmid containing the Pace promoter, but with a kanamycin
resistance) using the restriction enzymes BamHI/XbaI.
8. At this point, two fragments are visualized in agarose gel: a
3.2 kb and a 3.6 kb fragment. Because of the similar size of both
fragments, we recommend using a low-concentration agarose
gel to separate them. Purify only the 3.6 kb fragment.

Genetic Strategies on Kennedy Pathway to Improve Triacylglycerol Production. . .

137

9. Please note that atf2 gene (~1,380 bp) can be also subcloned
into pTip-QC2 vector with NdeI/HindIII from pGEM-T-easy/
atf2 directly. Alternatively, other restriction enzymes can be used.
10. Overnight incubation can also be performed with good results.
11. Wet biomass can be also used for a rapid analysis of neutral
lipids. For this, normalize cell pellets by OD600 measurements
of cellular cultures by triplicate.
12. The chosen amount of dry biomass depends on the expected
levels of TAG. For oleaginous strains, 5 mg is sufficient for TLC
assays as well as the quantitative analyses. For non-oleaginous
strains or assays under non-accumulation conditions, at least
10 mg of dry biomass is required.
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