Assay for Antibiotic Resistance

Steve HarrisSPASH – Biotech II

12/13/2010

Purpose

• To isolate β-lactamase from an overnight culture of ampicillin resistant E. coli

• To quantitatively explore the conversion penicillin to penicilloic acid.

• To explore factors that drive reaction rates.

12/13/2010

Protocol Day I: Pre-lab Preparation

• Prepare 25ml of .04% Phenol Red, 20mM Tris Solution– 1ml of 1% Phenol Red– 500μl of 1M Tris– 23.5 ml N-H2O

• Inoculate 200ml LB broth with E. coli – MM294

• Inoculate 200ml LB/AMP with E. coli –MM294+pAMP

• Incubate with shaking for 24hrs @ 37oc

.04%Phenol

Red

12/13/2010

LB LB + AMP

• Place 5ml E. coli into control tube.

• Place 5ml E. coli+pAMP into pAMP tube.

• Centrifuge 3min @6000rpm

• Decant supernatant into two new tubes.

Protocol Day II: Pre-lab Preparation12/13/2010

pAMPControl

ControlSup.

pAMPSup.

• Into a clear 1.5ml tube add– 700μl Penicillin-G– 100μl Phenol Red– 200μl Control Sup

• Into a clear 1.5ml tube add– 700μl Penicillin-G– 100μl Phenol Red– 200μl pAMP Sup

• Begin timing and observe color change to yellow.

• Record time of change in Data Table 1.

Protocol Day II: Timing of reaction

ControlSup

pAMPSup

pAMPSup

ControlSup

12/13/2010

ResultsData Table 1: Time of penicillin – G

conversion to Penicilloic Acid using Control Supernatant from E. coli + pAMP containing β-Lactamase

Time in minutes

Initial 0

Final

12/13/2010

Draw Chart in Lab Results

Data Table 1 : Absorbance of pAMP Supernatant from E. coli MM294 + pAMP

Time in Seconds

Absorbance @430nm

Time in Seconds

Absorbance @ 430nm

0 39030 42060 45090 480

120 510150 540180 570210 600240 630270 660300 690330 720360 750 (25min.)

Results 12/13/2010

Chart in Excel

Data Table 2: Mode of Action & Mechanism of Resistance - Homework:

Antibiotic Mode of Action Mechanism of Resistance

Ampicillin

A derivative of penicillin that kills cells by interfering

with a terminal reaction in bacterial cell wall

synthesis.

The resistance gene (amp or bla) specifies a periplasmic enzyme, β-lactamase, which cleaves theβ-lactam ring of the antibiotic.

Kanamycin

A bactericidal agent that binds to 70S ribosome's and causes misreading of

messenger RNA

The resistance gene (kan) specifies an enzyme (aminoglycoside

phosphotransferase) that modifies the antibiotic and prevents its interaction with ribosome's.

Tetracycline

A bacteriostatic agent that prevents bacterial protein synthesis by binding to the 30S subunit of ribosome's.

The resistance gene (tet) specifies a protein that modifies the bacterial

membrane and prevents transport of the antibiotic into the cell.

Results – Question #4

Spectral Analysis of pAMP Supernatant B-Lactamase Reaction

0

0.2

0.4

0.6

0.8

1

1.2

0 30 60 90 120

150

180

210

240

270

300

330

360

390

420

450

480

510

540

570

600

630

660

690

720

750

Time in Seconds

Abs

orba

nce

@43

0nm

12/13/2010

Graph in Excel

Mr. H. Messedwith our

experiment

Results – Question #5

Spectral Analysis of pAMP Supernatant B-Lactamase Reaction

0

0.2

0.4

0.6

0.8

1

1.2

0 30 60 90 120

150

180

210

240

270

300

330

360

390

420

450

480

510

540

570

600

630

660

690

720

750

Time in Seconds

Abs

orba

nce

@43

0nm

12/13/2010@

560n

m