Producing a Strain of E.coli that Glows in the

Dark

Exercise 10

Announcements

Post Lab 10 and Pre Lab 11 is due by your next lab

LNA Glow Lab is assigned today, and is due next week in lab

Exam II is during your lab period the week of April 24 and will cover Exercises 8-11

The Final Exam is scheduled for May 5 from 8-11 AM. Locations TBA. If this conflicts with any of your other final exams, you must fill out the Final Exam Conflict Request Form by 5 PM on May 3, 2017.

Goals

To create an ampicillin-resistant population of E. coli by introducing a plasmid that contains an ampicillin resistance gene

Understand the lux Operon and how it is used to create luminescent bacteria

Understand the pUC18 plasmid and how it serves as a control in this experiment

Penicillin

One of the most important anti-infective agents used in clinical medicine

Interferes with bacterial cell wall synthesis Over 500 semi-synthetic penicillins have been

made during the past 30 years

Plasmids

Plasmids are small circular DNA molecules that exist apart from the chromosomes in most bacterial species

Many contain genes that enable bacteria to survive and prosper in certain environments b/c they carry genes that provide resistance to antibiotics

pUC18 Plasmid

Circular DNA that contains 2686 nucleotides (very small)

Contains an ampicillin-resistance gene

pUC18 Plasmid

Process of introducing plasmids into living bacterial cells.

Procedure:•Treat bacteria with calcium

chloride

•Add the plasmid DNA

•Select for the transformed bacteria on

antibiotic media

Transformation

lux Operon

Contains two genes that code for luciferase and several genes that code for enzymes which produce luciferins

The lux Operon. The lux operon contains two genes for the luciferase enzyme (A and B). This enzyme is composed of two different polypeptide chains. The operon also contains several other genes (R, T, C, D, E, G, And Rib) that are thought to code for enzymes which produce the substrates for the light-emitting reaction. These substrates are called luciferins and are long chain fatty aldehydes.

The Exercise

Part I: Preparation of Competent Cells Review procedure on page 122 of your lab manual. This has

been done for you prior to starting the experiment.

Part II: Uptake of Plasmid by competent cellsPart III: Selection on ampicillin plates

Follow procedure in your lab manual. Note that to store your plates in the dark, you will put them in a paper sack and then in a box designated for your section

Your cells must stay on ice at all times !!!!

• Transfer 50 μL from the EC tube to each of the tubes labeled C (control), L (lux), and NP (no plasmid)

• Incubate tubes for 20 min on ice• Incubate tubes for 30 sec at 42°C• Incubate on ice for 5 min• Add 500 μL of Nutrient Broth to each tube and incubate at 37°C for 30

min at 230 rpm• Spread 100 μL of each tube into an agar plate containing ampicillin

(one plate per tube) (label plates)• Invert plates and incubate in dark

Protocol

Part IV:Examination of cultures in the dark

You will have to attend open hours on:

Results

Section Open Hour Time (210 Noyes)A Thursday (4/6); 9 AM – 3 PMB Thursday (4/6); 11 AM – 5 PMC Thursday (4/6); 2 PM – 8 PMD Thursday (4/6); 2 PM – 8 PME Thursday (4/6); 5 PM – 8 PM

Friday (4/7); 9 AM – 11 AMF Friday (4/7); 11 AM – 3 PMG Friday (4/7); 11 AM – 3 PMH Friday (4/7); 11 AM – 3 PM