SDS polyacrylamide gel electrophoresis (SDS PAGE)

Proteins were analysed on 10% slab gels as described by Laemmll

(1970). Gels were poured using a 'Biorad' 50 ml vertical gel apparatus. The

apparatus was assembled as described by the manufacturers. The 10% gel mix

was poured slowly between the glass plates using a 25 ml pipette until

approximately 5 cm from the top o f the plates. Butanol (H 20 saturated) was

then poured onto the top of the gel mix in order to ensure that the top of

the gel was level when polymerisation was complete (1-2 hr). The butanol

was then washed o ff by rinsing with H

20

before adding the stacking gel mix.

The stacking gel mix was added until it filled the gap between the top of

the running gel and the top of the glass plates. The well-forming comb was

instantly inserted into the stacking gel leaving a space of 3 cm between the

top o f the running gel and the bottom o f the wells. When set (20 min), the

comb was carefully removed and the wells washed out with running buffer

using a Pasteur pipette and teat. The gel was then transferred to the gel

tank containing running buffer and the samples loaded through the upper buffer chamber.

Items In [brackets] were added after de-gassing the solution (20 min

under vacuum).

Stacking gel buffer

Tris (0.5 M) was made by dissolving 5.98 g in 80 ml of H20 and then

adjusted to pH

6.8

before being made up to 100 ml with H

2

0.

Lower gel buffer

Tris (0.5 M) was made by dissolving 36.33 g in 80 ml of Hz O and then

adjusted to pH

8.8

before being made up to 100 ml with H

2

0.

Hl-bls acrylamide (low % gel) mix

A 60% stock was made by dissolving 60 g of acrylamide and 1.6 g of bis-

acrylamlde in 25 ml of H

2

0. The volume was later made up to 100 ml with

H

2

0 . This stock was stored at 4°C. 10% lower gel mix (50 ml)

8.3 ml hi-bisacrylamide

34.9 ml H20

6.25 ml lower gel buffer

0.5 ml 10% SDS

Stacking gel (10 ml)

3.0 ml stacking gel acrylamide

2.2 ml H20

2.4 ml stacking gel buffer

0.1 ml SDS [10 pi TEMED] [100 pi APS] [5 pi TEMED] [100 pi APS] 142-

Stacking gel acrylamide

10 g of acrylamide and 0.5 g of bis-acrylamlde were dissolved In a final

volume of H

2

O of 100 ml. This stock was stored at 4°C.

Running buffer

A 5 x concentrated stock was made by dissolving 30.2 g of Tris and 144 g

of glycine in H

2

O in a final volume of 1 1. Running buffer was prepared by

mixing 200 ml of this 5 x stock with 10 ml of 10% SDS and 780 ml of HzO.

Stacking gel acrylamide

10 g of acrylamide and 0.5 g of bis-acrylamide were dissolved in a final

volume of H

2

O of 100 ml. This stock was stored at 4°C.

Running buffer

A 5 x concentrated stock was made by dissolving 30.2 g o f Tris and 144 g

of glycine in H

2

O in a final volume o f 1 1. Running buffer was prepared by

mixing 200 ml o f this 5 x stock with 10 ml of 10% SDS and 780 ml of H

2

0.

3.1. Introduction

The generation of Erwinia spp. mutants defective in extracellular

enzyme production has been described previously (section 1.9.8.). Various

techniques have been used to generate such mutants including transposon and

chemical mutagenesis. Mutants of Ecc defective in extracellular enzyme

production have been generated using various transposons (Tn^, TnphoA and

TnlO) in this laboratory (unpublished). However, in this study the chemical

mutagen EMS was used. A brief discussion on the merits o f transposon and

chemical mutagenesis will be given. This will be followed by a description of

the approach taken in this study and a discussion on the classes o f Ecc

mutants isolated.

3.2. Mutagenesis - Transposons versus chemical mutagens

Transposons can be used to generate single and tagged mutations. This

is a useful feature for cloning mutated genes. However, a major disadvantage

is that transposons sometimes integrate into 'preferred sites' or 'hot-spots'

in the bacterial chromosome. This may lead to the inability to generate

mutations in some genes.- Mutations generated by transposons are caused by

an insertion event into a gene resulting in a gross disruption o f that gene. It

was not known at the onset of this study if transposon insertions Into genes

encoding extracellular enzyme secretion proteins would be lethal. The

lethality of mutations In the genes involved in the export apparatus of

E. coli ( sec genes) has been discussed (section 1.4.2.) and there was no

reason to suggest that the secretion apparatus might not also be part of a

general, essential process.

Chemical mutagens can be used to generate subtle and random

mutations by causing single base changes. Point mutations sometimes result in

conditional mutants which display a phenotype dependent on external factors

such as temperature. This technique has been used to isolate mutations in

essential processes including cell division and protein export in E. coll.

Mutants conditionally defective in extracellular enzyme production were

searched for in this study.

There are some disadvantages when using chemical mutagens. One

problem is the generation of multiple mutations. This can be reduced by

optimising the conditions of mutagenesis to produce a low but detectable

number of mutants. Mutations produced by chemicals (or UV light) cannot be

easily cloned, unlike transposon insertion mutants. Furthermore, chemical

mutagens are often highly toxic and carcinogenic.

3.3. The use of ethyl methyl sulphonate (EMS) to generate mutants of Bcc

In this study the chemical mutagen ethyl methyl sulphonate (EMS) was

used. The chemical formula of the EMS molecule is CH

3

SO

3

CH

2

CH

3

. This

chemical is a powerful alkylating agent and has been used previously to

generate mutations in Ecc SCRI193 (Forbes and Perombelon, 1985). EMS

alkylates the purine bases, adenine and guanine, which are subsequently lost

from the DNA (phosphodiester linked deoxyribose) backbone. During the next

round of DNA replication, DNA synthesised from this damaged template has

random nucleotides inserted in the positions where gaps in the template exist

3.4. Results

3.4.1. Construction of a killing curre

Ecc HC131 was grown and treated with EMS as described In section

2

.

6

.

Samples of bacteria were taken at 30 min intervals after the addition

of EMS. The viable count was determined at each time point and the

percentage survival as compared with the viable count before the addition of

EMS (t=0 min) was determined.

On the first run of this experiment, an exposure to EMS for 180 min

resulted in a survival of 0.4%. The killing curve was then repeated taking

frequent culture samples at

20

min intervals from between

100

and

220

min

after the addition of EMS. The results are illustrated in Figure 3.1.

A survival of between 1 and 4% was sought which was expected to

generate auxotrophs in the surviving culture at a frequency o f 1 to 4%

(Forbes and Perombelon.1985).

3.5. Survival o f Ecc after EMS treatment

A culture of Ecc was treated as described in section 2.6. under

conditions expected cause adequate mutagenesis, as determined from the

killing curve (Figure 3.1.). The survival o f a bacterial culture o f Ecc after

being treated with EMS for 90 min was found to be 3.5%. The survival is an

expression o f the viability of the culture a fter EMS treatment (t=90 min)

compared to the viability before ( t

=0

min).

This frequency of survival was within the limits used by Forbes and

Perombelon (1985). The mutagenised bacterial suspension was stored at 4°C

until required. The mutagenised cell suspension was serially diluted and plated

Figure 3.1. Survival of E c c treated

In document Molecular genetic analysis of extracellular enzyme secretion by Erwinia carotovora (Page 174-182)