Objetivos de materia

To determine whether disruption of efflux pumps altered lipopolysaccharide (LPS)

production, LPS was prepared and analysed. A 5 ml of culture of bacterial strains of

interest were grown in LB broth at 37°C overnight with shaking (200 rpm). The optical

density of the overnight culture were measure at 600 nm, ideally the overnight

cultures will have reached an optical density at 600nm of 3.5 or higher. 250 µl of the

overnight culture was dispensed into a microcentrifuge tube and pelleted via

centrifugation at 14,000 x g for 10 minutes. All supernatant was discarded and pellet

re-suspended in cracking buffer (SDS-Page loading buffer), the volume of which is

calculated by multiplying the optical density at 600nm of the overnight culture by 25

(e.g if the optical density at 600 nm of the overnight culture is 4, then 100 µl of

cracking buffer would be used to re-suspend the pellet). This suspension was then

boiled for 4 minutes and frozen at -80°C for 5 minutes. The frozen lysates were then

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centrifugation in a microcentrifuge at 14,000 x g for 2 minutes. Around 80 µl of the

supernatant was discarded and 5 µl of 5 mg/ml of Proteinase K to digest any protein

(A 5 ml/mg stock of Proteinase K was prepared by diluting a 20 mg/ml stock 1 in 4

with cracking buffer). This mixture was incubated at 60°C for 1 hour and then heated

to 95°C for 5 minutes. 4 µl of loading buffer was added to 10 µl of LPS sample heated to 95°C for 5 minutes and then loaded onto a 10% Bis-Tris Bolt™ gel (Life

Technologies, U.K.) and electrophoresed at 80 V for around 1 hour. Bis-Tris Bolt™

gels were stained using SimplyBlue™ SafeStain (Life Technologies, U.K.) (see

section 2.12.4).

2.12.2 Outer membrane protein preparation

3 ml overnight cultures of strains were prepared in LB broth and incubated at 37°C

with shaking (200 rpm). 50 ml of pre-warmed LB broth without salt was inoculated

with a 4% inoculums of the overnight culture and incubated at 30°C until mid to late

logarithmic phase is reached (an optical density at 600 nm of 0.7-1.0 for Salmonella).

Around 25 ml of this culture was transferred to a sterile universal tube and pelleted

via centrifugation at 3000 x g at 4 °C for 15 minutes. The supernatant was discarded

and the remainder of the culture transferred on top of the pellet and centrifugation

repeated. The supernatant was once again discarded and the pellet was

re-suspended in 10 ml of ice cold sterile PBS to wash cells. Pellets were again

obtained via centrifugation at 3000 x g at 4°C for 10 minutes. Samples were kept on

ice between steps. The supernatent was once again discarded and pellet

re-suspended in 1 ml of ice cold sterile PBS, the sample was then transferred to a

sterile glass bijou tube and stored at -20°C overnight. The following day samples

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four times at 30 seconds with a 30 second rest on ice. Sample was then transferred

to a labelled ice cold microcentrifuge tube and pelleted via centrifugation at 5,000 x g

at 4°C for 5 minutes. The supernatant was discarded and the pellet was

re-suspended by vortexing in 200 µl of a 2% sarcosyl solution and incubated at room

temperature for 30 minutes. The sample was once again pelleted via centrifugation at

10,000 x g at room temperature for 30 minutes. The supernatant was again

discarded and pellet air dried for 15 minutes to ensure the pellet is completely dry.

The dry pellet was re-suspended by vortexing in 50 µl of sterile PBS. The sample

was peletted via centrifugation in a microcentrifuge at room temperature at 10,000 x

g for 10 minutes to wash off any remaining sarcosyl. The supernatant was discared

for the final time and the pellet re-suspended in 50 µl of sterile PBS and can be

stored at -20°C prior to estimating the amount of protein present (see section 2.12.3) and analysing on a 10% Bis-tris Bolt™ gel (see section 2.12.4).

2.12.3 Bradford assay for protein quantification

A set of 5 bovine serum albumin (BSA) (Sigma, U.K.) standards were prepared; 0.1,

0.2,0.3, 0.4 and 0.5 mg/ml. Transfer 20 µl of each standard into clean microcentrifuge

tubes and add 1 ml of Bradford reagent (Sigma, U.K.) (Bradford 1976). Samples

were incubated at room temperature for 5 minutes. A no protein control (1 ml of

Bradford regent and 20 µl of water) in a cuvette was used to calibrate a

spectrophotometer at 595 nm. Each sample was transferred to a cuvette and optical

density at 595 nm was then measured in a spectrophotometer and plotted on a graph

of optical density against BSA concentration (Figure 2.5) on Microsoft Excel 2010

(Microsoft, USA). A line of best fit was added to the graph and equation of the

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Figure 2.5 A representative Bradford assay standard curve for protein quantification using standards of Bovine Serum Albumin (BSA)of 10, 20, 30, 40 and 50 µg/ml and corresponding optical density values at 595 nm plotted on a line graph. Line of best fit and equation of a straight line calculated using Microsoft Excel 2010 (Microsoft, US) and equation rearranged and annotated to allow estimation of unknown protein samples from their optical density value.

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20 µl of the protein sample to be quantified was added to 1 ml of Bradford reagent in

a cuvette and optical density at 595 nm measured on a spectrophotometer. To

calculate the quantity of protein in the sample put the optical density obtained into the

equation of the line of best fit from the graph. Each protein preparation was diluted

the match the protein sample of the lowest concentration before being analysed on a

10% Bis-tris Bolt gel (see section 2.12.4)

2.12.4 OMP/LPS gel staining

OMP and LPS Bolt™ gels were stained with SimplyBlue™ SafeStain (Invitrogen,

U.K.). Gels were transferred into a square petri dish and washed with 100 ml of

Ultrapure water with gentle agitation for 5 minutes. This wash step was repeated a

further 2 times. The gel was then stained with 20 ml of SimplyBlue SafeStain for 1

hour with gentle agitation. The stain was then removed and washed with 100 ml of

Ultra pure water with gentle agitation for 1 hour. Gels were visualised on a

transilluminator with a white light converter.