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Periplasmic expression in E. coli, ligand binding assays

 

In this block of the course you will learn the following methods:

Expression of recombinant proteins into E. coli periplasm

Ni-chelate chromatography of His-tagged proteins

Saturation and competitive binding experiments to analyze protein-ligand interactions

 

You will have to present this week’s results in form of a short scientific paper, which should include the following sections: Summary (max 80 words), Introduction (max 200 words), Results, and Conclusions. The report can be done either individually or in pairs. Please, send the report to Anton before January 11th.

 

The entire block will take place at in Biocenter (BioC)!

 

 

Lab 1: Expression to the E. coli periplasm

Lab 2: Determination of binding constants

Read this booklet on analyzing radioligand binding data

 

 

Start night cultures of the FimH-LD expressing cells on Sunday (Dee)!

 

Nov 21, Monday Prepare for a long day!

9:00-10:00 Wet lab: Expression to the E. coli periplasm: purification of lectin domain of FimH subunit (FimH-LD) of type 1 pili (Dee) BioC:Lab

Expression

10:00-11:00 Lecture: Adhesive pili: secretion, structure, and receptor binding (Anton) Room: BioC C216

11:00-12:00 Wet lab: Expression to the E. coli periplasm: purification of FimH-LD (Anton) BioC:Lab

Expression

12:00-14:00 Lunch

14:00-18:00 Wet lab: Expression to the E. coli periplasm: purification of FimH-LD (Dee) BioC:Lab

Extraction from the periplasm, SDS PAGE

 

Nov 22, Tuesday

9:00-12:00 Wet lab: Expression to the E. coli periplasm: purification of FimH-LD (Dee) BioC:Lab

Ni-chelate chromatography

12:00-14:00 Lunch

14:00-17:00 Wet lab: Expression to the E. coli periplasm: purification of FimH-LD (Dee) BioC:Lab

Ni-chelate chromatography, dialysis

 

Nov 23, Wednesday Prepare for a long day!

9:00-12:00 Wet lab: Expression to the E. coli periplasm: purification of FimH-LD (Dee) BioC:Lab

Gel filtration

12:00-14:00 Lunch

14:00-15:00 Lecture: Saturation and competitive binding experiments (Anton) Room: BioC C216

15:00-18:00 Wet lab: Expression to the E. coli periplasm: purification of FimH-LD (Dee) BioC:Lab

Gel filtration, SDS PAGE

 

Nov 24, Thursday

9:00-12:00 Wet lab: Determination of the binding constant between FimH-LD and mannose (Anton) BioC:Lab

Filter assay, beta-counter

12:00-14:00 Lunch

14:00-17:00 Determination of the binding constant between FimH-LD and mannose (Anton) BioC:Lab

Filter assay, beta-counter

 

 

Nov 25, Friday

 

9:00-13:00 Computer labs: Analysis of the binding data (Anton) Room: Bibliotekets Datorsal 2

 

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