1. Gene Regulation
pGLO Transformation

2. Gene Regulation Used for: Cell specialization Developmental changes
Adaptation to environment
Preventing overproduction of certain proteins

3. How are Genes Regulated?
By switching “on/off” transcription (DNA  mRNA)
Regulation happens at promoter or operator regions
Related genes are grouped together and use the same promoter and operator (Operons!)

4. The Arabinose Operon in E.coli
Genes code for enzymes used to breakdown arabinose (sugar)
Three genes control this: araB, araA and araD
Transcription of these genes requires:
DNA template (entire operon)
RNA Polymerase
DNA binding protein called araC
Arabinose to be present
Form single promoter called PBAD

5. 1) araC binds to DNA
2) arabinose interacts with araC to change its shape
3) Helps RNA Polymerase bind
4) Genes are transcribed  enzymes made to break down arabinose
As arabinose is used up, it breaks away from araC, shutting down transcription  another self-regulatory process!

6. The pGLO Plasmid Engineered to incorporate aspects of arabinose operon
Promoter (PBAD) and araC gene present
Genes for enzymes have been replaced with:
1) gene coding for GFP (Green Fluorescent Protein)
AND
2) gene that results in resistance to the antibiotic ampicillin (bla gene)
So… when arabinose is present, araC does its thing and the GFP is produced! Cells will fluoresce a brilliant green!

8. We need to induce the bacterial cells to “take up” the pGLO plasmid… We do this through a hot/cold shock treatment
Cells that take up the pGLO plasmid AND are provided with arabinose should express the GFP!

9. GFP gene replaces araB/ araA/ araD sequence
*Gene coding for ampicillin resistance (bla gene) is ALSO present with the GFP gene but isn’t linked to the araC/arabinose operon!
In the presence of arabinose, the GFP gene is expressed!

10. Setting up the Plates
Key: +pGLO = plates will be given the special plasmid -pGLO = plates will not have the special plasmid LB = simple nutrient agar amp = plate treated with antibiotic ampicillin ara = plate treated with arabinose

11. 4 Treatment Plates Expected Results? Plate #1:
Bacterial growth but NO fluorescing
Treated with pGLO plasmid but NO arabinose to “activate” the special plasmid (for fluorescing)
Contains extra bla gene for ampicillin resistance
+pGLO
LB/amp
Expected Results?
Plate #2:
Bacterial growth! Fluorescing colonies!
Treated with pGLO plasmid + ampicillin but has arabinose needed to activate it!
+pGLO
LB/amp/ara

12. 4 Treatment Plates Expected Results? Plate #3: No bacterial growth
Treated with ampicillin AND lacks the plasmid DNA
-pGLO
LB/amp
Expected Results?
Plate #4:
Even lawn of bacterial growth
No treatment apart from regular E.coli spread on nutrient agar
-pGLO LB