1. Unit 5 Day 3
Lab Results

2. Warm Up
Get out your lab procedure and turn to the back , where there are 4 petri dishes drawn.

3. Agenda
Lab results In depth look at the procedure Start lab report: due on Tuesday!!!!

4. Lab Results Look at your bacteria For the amount of growth!
If there is glowing! (use UV light)
Draw results in lab procedure
Return bacteria to incubator and wash your hands!!

5. Lab Report
Look at today’s ppt on website to get additional information (start on slide 6- we are in the genetics unit) Go to google classroom to see requirements for lab report and begin writing! DUE TUESDAY- assessment grade

6. Reasons for performing each transformation step
Ca++ O
CH2 P
Base OH
Sugar
Transformation solution = CaCl2
Positive charge of Ca+2 ions shields negative charge of DNA phosphates

7. Reasons, cont. Incubation on ice slows fluid cell membranes
Heat-shock increases permeability of cell membrane
Nutrient broth incubation allows beta lactamase expression
GFP
Cell wall
Beta lactamase
(ampicillin resistance)

8. Aequorea victoria

9. The Experiment
We are going to use a manufactured plasmid to attempt to transform E.Coli
Plasmid?
Small circular piece of DNA found in bacteria
Separate from main chromosome
Transform?

10. Plasmid genes? The plasmid contains The inducible ara operon
Three genes:
ara C: codes for repressor protein
GFP: codes for a protein that glows in the dark
bla: codes for a protein that provides resistance to ampicillin (an antibiotic)
pGLO
ori
bla
GFP
araC

11. Arabinose (ara) operon
When arabinose is present:
It binds to repressor
Turns gene on (repressor released from operator)
Transcription and translation take place
In the pGlo plasmid, the normal arabinose genes have been replaced (using gene splicing) with the GFP gene
Since arabinose is the co-repressor:
Arabinose present=glowing bacteria (if they have the plasmid)

12. Gene Regulation RNA Polymerase araC ara GFP Operon GFP Gene ara OperonB A D
Co-Repressor (Arabinose)
Co-Repressor (Arabinose)
araC B A D
RNA Polymerase
araC B A D