BME

MOLECULAR BIOLOGYEXPERIMENT TRANSFORMATION & SPREADINGSKKU BME

3RD GRADE, 2ND SEMESTER

PIPETTE MAN

• Caution!

• How much can you push?

• Upside down?

• Suck more and more?

BROTH FOR BACTERIA AND VIRUS

BROTH FOR MICROORGANISM

LYSOGENY BROTH

• 10 g tryptone

• 5 g yeast extract

• 10 g NaCl

Why???

HOW CAN YOU SELECT ONE TYPE OF E. COLI?

TWO WAYS

• Antibiotics

• Colony

THEN HOW??

Antibiotics-resistance

X X X XX XX XX X

PENICILLIN

Alexander Fleming

ANTIBIOTICS

• Ampicillin & Penicillin

Β-LACTAM ANTIBIOTICS

• β-lactam antibiotics (beta-lactam antibiotics) are a class of broad-spectrum antibiotics, consisting of all antibiotic agents that contain a beta-lactam ring in their molecular structures.

WHY DO YOU KNOW THE MECHANISMS OF CHEMICALS AND PROTEINS IN MOLECULAR BIOLOGY EXPERIMENT?

• You can see as much as you know.

• For making a breakthrough

• For trouble shooting

ANTIBIOTICS MECHANISM

THEN, WHAT SHOULD YOU DO?

• LB

• Antibiotics

• Then?Live or die?

THEN, WHAT SHOULD YOU DO?

• LB

• Antibiotics

• Then?Something for antibotics-resistance

NOW, WHAT DO WE WANT TO DO?INSERT THE DNA INTO THE E. COLI!!!

• Transformation + 2 more ways (H.W.)

TRANSFORMATION

OK! You can use this transformation in lab.

Basic key question:Why is this transformation necessary for E. coli?

TRANSFORMATION

How to insert the plasmid into the cell?

Hint>What’s the barrier of E. coli?How to make it permeable?

TWO WAYS OF TRANSFORMATION

IMAGINE

THINK THE PROBLEMS!!!

AFTER HEAT SHOCK TRANSFORMATION

• Seed this in the LB + antibiotics èWhat do you expect?

AFTER HEAT SHOCK TRANSFORMATION

• Seed this in the LB + antibiotics èWhat do you expect?

AFTER HEAT SHOCK TRANSFORMATION

• Seed this in the LB + antibiotics èWhat do you expect?

• Culture without antibiotics for a while…

NEXT PROBLEM – CONTAMINATION?OR NON-WANTED PLASMID?

• How to select one?

One plasmid in one E. coli group!!!

HOW TO SEPARATE EACH PLASMID IN E.COLI?

• One plasmid in one E. coli

• Then?

• Low concentration culture?

• How to pick up one plasmid-transformed E. coli?

THINK YOU SPREAD LOW CONCENTRATION OF E.COLI ON THE LA PLATE…

IMAGINE

START EXPERIMENTMaking broth

Transformation

Spreading E. coli

LET’S MAKE THE BROTH FIRST!

BROTH FOR MICROORGANISM

• Lysogeny broth (LB)

• Lysogeny + Ampicillin

• Lysogeny + Kanamycin…

• Selection broths

PREPARATION FOR THE BROTH

• 10 g tryptone

• 5 g yeast extract

• 10 g NaCl

• Clean water

• Mass cylinder

• Flask

• Petri dish

• Autoclave

• Aluminum foil

• Autoclave tape

PREPARATION FOR TRANSFORMATION!

• Competent cell

• Plasmid DNA

• Selection marker

• Transformation media

• Heat block

• Water bath

PREPARATION FOR SPREADING

• Transformed cell

• LB + antibiotics agar plate

• Spreader

CLEAN BENCH

• BUT

• You can use magic spells!!

CLEAN!!!!

PREPARATION FOR SPREADING

• Transformed cell

• LB + antibiotics agar plate

• Spreader

• Alcohol lamps

HOW ABOUT VIRUS?

Max D.

HOMEWORK

• Why are the tryptone, yeast extract and NaCl necessary for LB?

• How does microorganism get the antibiotics-resistance? Explain the gene and mechanism.

• Find three ways of gene transfer in E. coli.

• Please find the ampicillin-resistance gene and explain its mechanism.

• Who is Max Delbruck (virus counting method)?

• How to culture the influenza virus? Which broth, media and host are necessary?

• What are the critical factors to make a primer for PCR?

• What is the annealing temperature in PCR? How to calculate it?

NEXT WEEK

• PCR of template

• Vector seeding