Is taking in a plasmid good or bad for the bacteria? Explain. What will happen to the bacteria in the two types of growth media? Consider cells with or without plasmid. LB – Agar medium 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 rfp Rfp protein arabinose rfp

Lab 5a Conclusions 1: Expectations Observations

Recombinant plasmid of interest pARA-R construct Recombinant plasmid of interest pARA-R 4720 bp pARA-R 4720 bp BamH I rfp 702bp rfp 702bp Hind III 8

growth of transformed bacteria on various plates P+ plates LB LB/amp LB/amp/ara P- plates No growth LB LB/amp 9

Colony isolation and culture preparing an overnight culture of E. Coli for RFP expression Colony isolation and culture LB/amp/ara broth 11

Accordingly – As a group – make predictions on bacterial growth on 3 different plates: LB LB-Amp -Arabinose LB-Amp

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