Short Report2

8/6/2019 Short Report2

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Short report

Material and Methods/ Results

DNA methods

Transformation

Minipreps

Enzymatic digestions

Maxipreps

Transformation of expression strains

Protein methods

Whole organism Drosophila

Protein extraction method

Gel electrophoresis

Imunoblotting

Bacterial strains

Protein expression induction

Protein purification

Affinity chromatography


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DNA methods

Transformation

The plasmids pET30-hsp22 and pET30-hsp22 were transformed into the E.coli

strain DH5, which is one of the hosts for cloning according to the pET System Manual

from Novagen. A rapid transformation protocol, which consists in thawing a 100 l

aliquot of the DH5 competent cells on ice, adding 1 l of the plasmid, incubating the

tubes on ice for 20 minutes, heat pulsing then in a 42C water bath for 45 seconds and

reincubating them on ice for 2 minutes was used. After that 900 l of Soc medium were

added and incubated at 37C for 30 minutes with shaking at 225 250 rpm. At this point

LBagar Kanamicin plates had been prepared. 100 l were plated into one plate using a

sterile spreader and the remaining 900 l were plated in another plate. These were

incubated at 37C overnight.

Minipreps

Using the plates resulting from the previous step, one single colony was isolated,

inoculated into 2 ml of LB Kanamicin and allowed to grow at 37C overnight. It was

done for both plasmids, with 4 replicates (from 4 different colonies) each.

An alkaline lysis protocol was used to extract the plasmids from the cells and a

1% agarose gel electrophoresis was performed (the gel was stained with Etidium

Bromide). (Figure 1).


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Some plasmid extractions were also done with the QIAprep Spin Miniprep Kit

Protocol using a micro centrifuge, from Qiagen, because the DNA resulting from the

Alkaline Lysis method seemed to resist to digestion with restriction enzymes.

Enzymatic Digestions

The DNA was first digested with the enzyme XhoI in a total volume of 50 l (at

this step 20 g/ml of RNAse were added). The enzymatic reaction was incubated at 37C

for 2 hours. Then 20 l were used to do a 1% agarose gel electrophoresis (Figure 2). The

remaining DNA was precipitated with ethanol and ressuspended it in 20 l of water

before the second digestion, this time with the enzyme HindIII. This step was necessary

because these enzymes have different buffers, which means the second enzyme couldntbe mixed with the first reaction. The second digestion was also made in a total volume of

50 l, and incubated at 37C for 2 hours. After this a 1% agarose gel electrophoresis was

made (Figure 3).

A B C D M E F G H

Figure 1: Agarose Gel Electrophoresis. Lanes A, B, C and D have thefour replicates of pET30-22 transformed into DH5. Lanes E, F, G and F have

the four replicates of pET30-27 transformed into DH5, and M is the lanewith a 1 Kb marker.


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A B C D M E F G HFigure 2: Agarose Gel Electrophoresis of the DNA digested with

XhoI. Lanes A, B, C and D have the four replicates of pET30-22. Lanes E, F,

G and F have the four replicates of pET30-27, and M is the lane with a 1 Kb

DNA Ladder (Gibco).

B C D M E F G H

Figure 3: Agarose Gel Electrophoresis of the DNA digested with XhoI

and HindIII. Lanes A, B, C and D have the four replicates of pET30-22. LanesE, F, G and F have the four replicates of pET30-27, and M is the lane with a 1

Kb DNA Ladder (Gibco).


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It seems like there was no second digestion, which makes sense if one looks at the

pET30 system map (Novagen) in Appendix I. If indeed the cDNAs were inserted

between the NcoI and the XhoI sites, the HindIII site was gone, and none of the inserts

has one. Based on that map several digestions were planned:

1. 2 hours digestion with XhoI (37C) followed by a 2 hours digestion with

BglII (37C). This digestion should cut of the insert in both plasmids. The approximate

expected sizes would be:

pET30-hsp27 5400

600

pET30-hsp22 5400

500

2. 2 hours digestion with XhoI (37C) followed by a 2 hours digestion with

NcoI (37C). This digestion should cut of the insert only in pET30-hsp27. The

approximate expected sizes would be:

pET30-hsp27 5400

600

pET30-hsp22 5900

3. 2 hours digestion with SmaI (25C). This digestion should cut pET30-

hsp22 once and pET30-hsp27 twice. The approximate expected sizes would be:

pET30-hsp27 4400

1600

pET30-hsp22 5900

4. 2 hours digestion with EcoNI (37C). This digestion should cut pET30-

hsp22 twice and pET30-hsp27 three times. The approximate expected sizes would be:

pET30-hsp27 3700

2000

300


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pET30-hsp22 3700

2200

Figure 4 shows the result of these digestions in a 1% agarose gel electrophoresis.

For some reason the DNA didnt behave like expected. The decision was made to

proceed with the Maxi prep anyway.

Maxi prep

One colony of the transformed cells was isolated, inoculated into 2 ml of LB

Kanamicin and allowed to grow for about 16 hours at 37C, both for the DH5-pET30-

hsp22 and for the DH5-pET30-hsp27. Afterwards, these pre-inoculums were inoculated

A B C D E M F G H I J M

Figure 4: Agarose Gel Electrophoresis of the DNA digested with

different restriction enzymes. Lane A pET30-22 digested with XhoI andBglII; Lane B pET30-22 digested with XhoI and NcoI; Lane C pET30-22

digested with SmaI; Lane D pET30-22 digested with EcoNI; Lane E nondigested pET30-22; Lane F pET30-27 digested with XhoI and BglII; Lane G pET30-27 digested with XhoI and NcoI; Lane H pET30-27 digested with

SmaI; Lane I pET30-27 digested with EcoNI; Lane J non digested pET30-

27 and M is the lane with a 1 Kb DNA Ladder (Gibco).


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into 1L of LB-Kanamicin and incubated overnight at 37C. The DNA was purified

according to Promegas Technical Bulletin. The concentration of the purified DNA was

calculated using its absorbance at 260 nm, and its purity was measured by the

(Absorbance at 260)/(Absorbance at 280) nm ratio. Hsp27 was at a concentration of 30.6

mg/ml and hsp22s concentration was 8.43mg/ml. They were considered pure, since the

(Absorbance at 260)/(Absorbance at 280) nm was approximately 1.80 in both.

Transformation into expression strains

The strains BL21DE3 and GJ1158 were made competent and transformed in the

same way as described for DH5, except that GJ1158 had to be plated in LBONagar-

Kanamicin instead of LBagar-Kanamicin. DNA Minipreps were performed before

inducing the protein expression, and a 1% agarose gel electrophoresis was made(Figure

5)

Whole organism Drosophila

M A B C D E F

Figure 5: Agarose Gel Electrophoresis. Lane A

pET30-hsp22 from the maxiprep; Lane B pET30-

hsp27 from the strain GJ1158; Lane C pET30-hsp22from the strain BL21DE3; Lane D pET30-hsp27 from

the maxiprep; Lane E pET30-hsp22 from the strainGJ1158; Lane F pET30-hsp27 from the strainBL21DE3. M is a molecular weight ladder ( Hind III

Gibco)


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Protein extraction method

Approximately 70 flies were stressed by placing them inside 50 ml Falcon tubes

in a water bath at 29C for 20 minutes and then at 36C for 100 minutes. Half of that

sample was given a recovery time of about 16 hours. They were all frozen in liquid

nitrogen until the moment of extraction. The control sample was frozen without being

exposed to the stress conditions.

The samples were added 500 l of 1% SDS , boiled during 5 minutes,

homogenized in a Potter device, boiled for another 5 minutes and briefly centrifuged.

The amount of protein in the samples was calculated by using the BCA method

(BCA protein assay kit - Pierce).

A 15% SDS-PAGE was then done, and loading 20 g/l of protein in each lane.

The run was done in a Biorad Mini-protean device. Part of this gel was dyed with

Coomassie Blue and the rest was transferred into a nitrocellulose transfer membrane.

An imunoblotting assay was performed with these membranes using the hsp22

and the hsp27 antibodies that youve sent (primary antibody); anti mouse IgG

alkaline Phosphatase conjugate (Sigma) and monoclonal anti rabbit imunoglobulins

Figure 6: SDS-PAGE (15%) of protein extracts from

D. melanogaster stained with Coomassie Blue. Lane A: heatshocked sample; lane B: heat shocked sample with 16 h

recovery; lane C: non heat shocked sample; Lane D is a

positive control (S2 heat shocked, sent by you) and M is a lanewith a molecular weight marker (Kaleidoscope Prestained

standards - Biorad)

A B C M D


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alkaline Phosphatase conjugate (Sigma) were used as secondary antibodies, the last step

being done with BCIP/NBT color development substrate (Promega).

Bacterial strains

Protein expression induction

One isolated colony of GJ1158-pet30-hsp22 and another one of GJ1158-pet30-

hsp27 were inoculated in 2 ml of LBON-Kan each; BL21-pet30-hsp22 and BL21-pet30-

hsp27 were also inoculated in 2 ml of LB-kan. These were all grown at 37C overnight.

Afterwords, these pre-inoculum were poured into 100 ml of fresh KB-Kan or LBON-

Kan, depending on the strain. They were incubated at 37C until the OD at 600 nm had

reached 0.6. The protein induction was performed as recommended in the protocol

youve sent us. The cells were then centrifuged at 3000g for 10 minutes, and the pellet

was ressuspended in 5 ml of a 1% SDS solution. The samples were boiled during 5

minutes, sonicated, boiled again for another 5 minutes and briefly centrifuged. One

microliter of each of these samples was loaded into a 15 % SDS-PAGE, to check if the

induction was working. Part of this gel was dyed with Coomassie Blue (Figure 8) and the

rest of it was transferred into a nitrocellulose transfer membrane. This membrane was

then used in an imunoblotting assay, with the antibodies -hsp27 and -hsp22 (Figure 9)

Figure 7: Imunoblot from D. melanogaster homogenates. a) Probing

with -hsp27 antibody; b) Probing with -hsp22 antibody; Lane A: heat

shocked sample; lane B: heat shocked sample with 16 h recovery time; lane C:non heat shocked sample; Lane D is a positive control (S2 heat shocked, sent

by you) and M is a lane with a molecular weight marker (KaleidoscopePrestained standards - Biorad).

a

M D C B A

b

M A B C D


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After realising the induction had been successful, the induction was repeated at a

larger scale, with 500 ml of culture medium.

Protein purification

All the purification steps were performed at 4 C, in a refrigerator, according to

the protocol youve sent. Fractions were collected from all chromatography steps, and a

Figure 8: SDS-PAGE (15 %) of cell extracts resulting from the

induction of protein expression. Lane M: molecular weight marker(Kaleidoscope Prestained standards - Biorad); Lane A: GJ1158 transformed

with pet30-hsp22; Lane B: BL21 DE3 transformed with pet30-hsp22; Lane C:GJ1158 transformed with pet30-hsp27.

M A B C

Figure 9: Imunoblot of the samples resulting from protein expression

induction a) Probing with -hsp27 antibody; b) Probing with -hsp22antibody; Lane A: GJ1158-pet30-hsp27 without induction; Lane B: BL21

DE3-pet30-hsp22 without induction; Lane C: GJ1158-pet30-hsp27 induced

with NaCl; Lane D: GJ1158-pet30-hsp22 induced with NaCl; Lane E: BL21DE3-pet30-hsp27 induced with IPTG; Lane F: BL21 DE3-pet30-hsp22

induced with IPTG and M is a lane with a molecular weight marker

(Kaleidoscope Prestained standards - Biorad).

a b

A B C D E F M A B C D E F


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SDS-PAGE (12%)was performed and the resulting gels were dyed with Coomassie Blue.

The gels were scanned in GS710 calibrated imaging densitometer Biorad and the

images treated with the Quantity one 4.2.1 software. The following images are

examples of the gels obtained form the different samples.

a)

b)

A B C D E F G M


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c)

d)

A B C D E F G M

A B C D E F G M


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Figure 11: 2D PAGE of the eluted fractions, dyed with silver stain. a)

GJ1158-pet30-hsp22; b) GJ1158-pet30-hsp27. At the right you can see themolecular weight marker (Low molecular weight marker - Amersham)

b)


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Affinity chromatography

We are now adapting two affinity chromatography protocols in order to find out

which proteins interact with hsp22 and hsp27. One of the protocols uses the same buffers

as the purification. The protein is bound to the resin, washed, Drosophilas homogenate

passed through the column, washed and eluted. The other approach uses AC buffer (10%

glicerol, 100mM NaCl, 20mM Tris pH 7.6, 0.5mM EDTA, 0.1 % Tween) more or less in

the same way. The chromatographies done so far dont show a big amount of protein in

the eluted fraction (Figure 13)

Figure 12: 2D PAGE of the re-purified eluted fraction of GJ1158-

pet30-hsp22, dyed with silver stain. At the right you can see the molecular

weight marker (Low molecular weight marker - Amersham)

Figure 13: SDS PAGE(12%) of all the fractions resulting from theaffinity chromatography, stained with Coomassie Blue. Lane A: Cell lysate;

Lane B: Flow throught; Lane C: Wash with Binding Buffer; Lane D: Wash

with Washing Buffer ; Lane E: Drosophila homogenate; Lane F: Flowthrough; Lane G: wash with Washing Buffer; Lane H: elution; Lane I: wash

with Strip Buffer and M is a molecular weight marker (prestained low range

A B C D E F G H I M

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