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1 DNA Amplification, Cloning, and Sequencing

1 DNA Amplification, Cloning, and Sequencing

Tải bản đầy đủ - 249trang

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



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



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



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



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



132



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.



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



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).



134



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).



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



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.

References

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lipid inclusion by Rhodococcus opacus PD630.

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3. Alvarez HM, Kalscheuer R, Steinb€

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