LB – Agar medium

1. Is taking in a plasmid good or bad for the bacteria? Explain.

2. What will happen to the bacteria in the two types of growth media? Consider cells with or without plasmid.

LB – Agar +

Ampicillin

Transformation - Animation

Lab 5a Student Guide

LB agar: LB = Luria-Bertani (lysogeny) Broth and it contains:10g of tryptone, 5g of yeast extract and 10g of NaCl.

Agar is the crude version of agarose, the gel we used for separation of DNA fragments.

Background before predictions:Arabinose operon:

Arabinose is a small sugar (monosaccharide), and not very common. When it binds AraC protein, DNA stays open (not looped) and allows RNA polymerase to transcribe the gene downstream.

See page 6.1

AraC dimer

arabinose

rfp

Rfpprotein

rfp

Lab 5a Conclusions 1: Expectations

Lab 5a Conclusions 2:Observations

pARA-R construct

Recombinant plasmid of interest

pARA-R

BamH I

Hind III

rfp702bp

4720 bp

rfp 702bp

pARA-R4720 bp

growth of transformed bacteria on various plates

P+ plates

P- plates

LB LB/amp LB/amp/ara

LB LB/amp

No growth

Colony isolation and culture

preparing an overnight culture of E. Coli for RFP expression

LB/amp/arabroth

Accordingly – As a group – make predictions on bacterial growth on 3 different plates:

LB LB-Amp LB-Amp-Arabinose

F- F+F- F+ F+

Lab Report:

Plate Predict-ions

Reason Results Why

LB

LB-Amp

LB-Amp-Ara

Sterile technique:To prevent contamination - •Minimize contact with skin. •Shorten open time.•Change tips, spreader. •Use ‘BioHazard’ bags.

“Clam Shell” technique:

LB-Amp

LB-Amp-Ara