Lecture 06

Transformation, Minimum Inhibitory Concentration (MIC), Siderophores

What is bacterial horizontal gene transfer?

Intact gene transfer from the same or different bacterial species

What is bacterial transformation?

• A stable genetic change, brought about by the uptake of exogenous naked DNA

What is does inducing competence refer to?

• Changing the cell in such a way that enables DNA to easily travel through the cell membrane

• E. coli, artificial cell competence is made possible chemically or through electroporation, creating temporary pores that allow DNA to enter the cell.

For chemically induced competence ( CaCl2 and heat-shock), what does ice and heat shock do?

• Ice: helps DNA adhere to surface & CaCl 2: promotes DNA binding and pore formation

• Heat shock: membranes more fluid and some DNA moves through membrane

• Cold temperature help membrane regain rigidity

What are ampR and GFP?

Marker genes

What happens to ampR (bla) and araC?

They are constitutively expressed

GFP expression is regulated by what?

AraC promoter

In GFP regulation, what does AraC control?

The promoter, P_BAD is fused to GFP

What is the standard protocol for E. coli transformation with pGlo?

1. Induce chemical competence by washing E. coli cells mid-log (during exponential growth) with CaCl₂

2. Place in ice

3. Add 10 ul pGLO plasmid to +DNA tube. Incubate on ice 10 min

4. Heat shock for 50 sec

5. Ice for 2 min

6. Add LB to each tube. Incubate at 37^oC for 30 min

7. Transfer and spread 100ul onto selective plates

What do you expect from the following plates:

What is transformation efficiency?

• Number of cells transformed per 1 micro-gram of DNA

• TE = number of transformants/amount of DNA used

• Shock temperature, incubation, plasmid size…etc will influence TE

How will we determine the MIC of Kanamycin for a planktonic culture of Pseudomonas?

• Prepare 2-fold dilutions of an antibiotic

• Inoculate with bacterium

• Incubate 24hrs

What is Pyoverdine?

• High Fe(III) affinity fluorescent siderophore, produced by Pseudomonas sp.

• Pyoverdine production is regulated by soluble iron available in environment

What are siderophores?

• Siderophores are synthesized then secreted outside the cell

• Chelate the insoluble iron and transport into cells via receptors

• Iron released inside the cell in a soluble form

What are sigma factors?

• Sigma factors provide promoter recognition specificity to RNA polymerase holoenzyme

• Sigma factors can provide effective mechanisms for simultaneously regulating expression of large numbers of prokaryotic genes

How is pyoverdine biosynthesis regulated?

• The pvdS sigma factor is required for the expression of the PVD operon

• Ferric uptake regulator (Fur); repressor protein inducible by iron

• Fur-controls pvdS gene (sigma factor) expression, required for the biosynthesis of pyoverdine

How is pyoverdine produced and regulated?

• High affinity and low affinity siderophores

• Pyoverdine is a high affinity siderophore

• Can strip Fe away from Dipyridyl

What is competing via siderophores?

• Secreted siderophores can be used by other strains that DO NOT make their own siderophores

• These strains are called pirates or
  social cheaters

How do you identify a cheater?

• Pyoverdine producers and non-producer strains

• Which Pvd - strain(s) can use pyoverdine?

  • Spread strains on CAA agar + dipyridyl

  • Add a source of PVD on top of overlay

  • PVD: CFS of strain A, grown in CA-broth