1. Preparing an Overnight Culture of Escherichia coli
Laboratory 6

2. Overview: Purpose: From LB/amp/ara plate:
Start a bacterial culture that will produce the mutant fluorescent protein
This will lead to the isolation and purification of that protein
From LB/amp/ara plate:
Will select one red colony that has been transformed with pARA-R plasmid
Inoculate an overnight culture

4. Introduction Gene for mFP originally isolated from sea anemone
Protein can fuse to other proteins – used in research
pARA-R major control elements:
Required to express the rfp gene
1. araC gene
Codes for AraC protein, which helps turn rfp gene on and off
Acts as a repressor by blocking promoter
2. promoter region (PBAD)
Portion of DNA where regulation of rfp expression occurs
RNA polymerase binds
3. rfp location

5. RFP expression Bruce Wallace araC gene PBAD rfp gene Transcription
mRNA
Translation
araC protein

6. Introduction 2. promoter region (PBAD) If no arabinose:
AraC protein physically bind to plasmid
At promoter site
At region near the araC gene
DNA bends around and blocks transcription cannot occur
RNA polymerase cannot bind to promoter site
Bacterium cannot produce mFP

7. RFP expression Bruce Wallace
araC protein prevents RFP transcription by causing
a loop to form in the region of the fp gene r
araC gene
araC protein
PBAD
rfp gene

8. Introduction 2. promoter region (PBAD) If arabinose present:
Arabinose binds with AraC protein
Prevents DNA loop from forming
RNA polymerase can join complex after conformational change
Three molecule complex binds promoter site on DNA
DNA transcribes rfp gene to produce mRNA
Translates to mFP!!
Stored as inclusion bodies

9. arabinose – araC protein
RFP expression
Bruce Wallace
RFP
(red fluorescent
protein)
Arabinose – araC protein complex prevents DNA looping
and helps to align RNA polymerase
on the promoter site (PBAD).
arabinose
Translation
RNA polymerase
araC protein
arabinose – araC protein
complex
mRNA
Transcription
araC gene
rfp gene
PBAD

10. Materials Materials: Equipment & Supplies:
LB/amp/ara plate (from Lab 5)
Sterile flask with LB/amp/ara broth
Equipment & Supplies:
Shaker
Plastic inoculating loops

11. What will you need to do?
Tell students to work quickly to avoid contamination
Discuss in detail rfp expression
AraC protein involved with turning arabinose operon ON and OFF!!
Binds to promoter OR arabinose
Need 2.5 mL overnight culture for each group
Use sterile loop to transfer small sample of cells to LB/amp/ara broth
Shake culture 1-2 days at room temperature

12. Methods Use a clean plastic inoculating loop to transfer a few cells
From mFP-expressing colony (red)
Into flask containing LB/amp/ara broth
Flask placed on shaker
Shaken overnight
Encourages cell division and rfp expression

13. Colony isolation and culture
Preparing an overnight culture of E. Coli for RFP expression
Bruce Wallace
Colony isolation and culture
LB/amp/ara
broth

14. Conclusions Cells grown that have been expressing mFP
Ready to be lysed and purified!