Team:TU-Delft/Protocol 9

Our project deals with E.coli cells which sense Auto-inducing peptides (AIPs) from the Staphylococcus aureus and starts producing Antimicrobial peptides in order to kill the Staphylococcus aureus. Different protocols used during the project are described below.

• Transforming Parts from Distribution kit
• Growing the Single Colonies from the Agar Plates
• Making glycerol stocks
• Plasmid Purification Protocol
• Restriction digestion
• Ligation
• Gel Extraction Procedure
• PCR Purification
• Tricine Gels
• General Peptide Production
• SUMO cleavage
• Lysis Protocol
• AIP Sensing Protocol
• Gene Design
• Primer Design

Tricine Gels

Tricine Gels [1] are ideal for peptides and low molecular weight proteins (less than 10 kDa). The Tricine Gels are based on the Tricine system developed by (Schaegger and vonJagow, 1987). In this buffer system, tricine substitutes glycine in the running buffer resulting in more efficient stacking and destacking of low molecular weight proteins and higher resolution of smaller peptides. Tricine gels must be used with denatured or reduced proteins only. The separating range of Tricine gels is 2.5-200 kDa.

Requirements:

1. Protein sample
2. Deionized water
3. Low molecular weight Protein markers
4. Tricine SDS Sample Buffer
5. Reducing Agent for reduced samples (Optional)
6. Tricine SDS Running Buffer

Preparing samples

The Tricine SDS Sample Buffer (2X) and Reducing Agent (10X) are available from Life Technologies (Invitrogen).

1. Prepare reduced or non-reduced samples for Tricine gels as described below:

Note:For reduced sample, add the reducing agent immediately prior to electrophoresis to obtain the best results.

Reagent	Reduced Sample	Non-reduced Sample
Sample	x µL	x µL
Tricine SDS Sample Buffer (2X)
Reducing Agent (10X)	1 µL	-
Deionized Water	to 4 µL	to 5 µL
Total Volume	10 µL	10 µL



2. Heat samples at 85℃ for 2 minutes. Load the sample immediately on the gel.

Preparing Running Buffer

1. Prepare 1000 ml of 1X Tricine SDS Running Buffer using Tricine SDS Running Buffer (10X) as follows:
   

   Tricine SDS Running Buffer (10X)	100 ml
   Deionized Water	900 ml
   Total Volume	1000 ml

2. Mix thoroughly. Use this buffer to fill the Upper and Lower Buffer Chambers of the XCell SureLock. Mini-Cell for electrophoresis.

Tricine SDS Sample Buffer (2X)

450 mM Tris HCl
12% Glycerol
4% SDS
0.0025% Coomassie® Blue G
0.0025% Phenol Red
pH 8.45

1. To prepare 10 ml of 2X Tricine SDS Sample Buffer, mix the following reagents:

   3 M Tris HCl, pH 8.45	3 ml
   Glycerol	2.4 ml
   SDS	1000 ml
   0.1% Coomassie® Blue G	0.5 ml
   0.1% Phenol Red	0.5 ml

2. Mix well and adjust the volume to 10 ml with ultrapure water.
3. . Store at +4℃. The buffer is stable for 6 months when stored at +4℃.

Tricine SDS Running Buffer (10X)

The Tricine SDS Running Buffer is available from Life Technologies (Invitrogen).
100 mMTris base
100 mM Tricine
0.1% SDS
pH 8.3

1. To prepare 1000 ml of 10 X Tricine SDS Running Buffer, dissolve the following reagents in 900 ml deionized water:
   

   Tris Base	3 ml
   Glycerol	121g
   Tricine	179g
   SDS	10g

2. Mix well and adjust the volume to 1000 ml with ultrapure water.
3. Store at room temperature. The buffer is stable for 6 months when stored at room temperature.
4. For electrophoresis, dilute this buffer to 1X with water. The pH of the 1X solution is 8.3. Do not use acid or base to adjust the pH.

Electrophoresis Conditions

Instructions for running the Tricine gel:

SimplyBlue SafeStain Protocol

The staining of the gels was done using SimplyBlue Safe Stain because the process of fixing the gel is avoided.
After electrophoresis follow the instructions below. Be sure the mini-gel moves freely in water or stain to facilitate diffusion during all steps.

1. Rinsethe mini-gel 3 times for 5 minutes with 100 ml deionized water to remove SDS and buffer salts, which interfere with binding of the dye to the protein. Discard each rinse.
2. Stain the mini-gel with enough SimplyBlue. SafeStain (20-100 ml) to cover the gel. Stain for 1 hour at room temperature with gentle shaking. Bands will begin to develop within minutes. After incubation, discard the stain. Stain cannot be re-used.
   Note: Gel can be stained for up to 3 hours, but after 3 hours, sensitivity will decrease. If you need to leave the gel overnight in the stain, add 2 ml of 20% NaCl (w/v) in water for every 20 ml of stain. This procedure will not affect sensitivity.
3. Wash the mini-gel with 100 ml of water for 1-3 hours. The gel can be left in the water for several days without loss of sensitivity. There is a small amount of dye in the water that is in equilibrium with the dye bound to the protein, so proteins will remain blue.
4. To obtain the clearest background for photography, perform a second 1 hour wash with 100 ml water.
   Note: Sensitivity will now decrease if the gel is allowed to stay in the water more than 1 day. Reduction of free dye in the water favors dissociation of the dye from the protein. If you need to store the gel in water for a few days, add 20 ml of 20% NaCl.

References

1. Novex Pre-Cast Gel, Electrophoresis guide, Version B January 27, 2003 IM-1002..[Online]. Available From: http://kirschner.med.harvard.edu/files/protocols/Invitrogen_novexelectrophoresisguide_man.pdf.