Team:TU-Delft/Protocol 12

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

Lysis Protocol

Procedure

1. Grow the BL21(DE3) cells transformed with pT7 lysis cassette overnight in LB meduim with the right antibiotic, (in this case ampicillin or carbenicillin) .
2. Make a 10X stock solution of IPTG by adding 10µL of the 1000X IPTG (1M) in 990 µL of LB with the right antibiotic.
3. Make 1/50 times dilutions of the overnight grown culture. Wait untill the OD at 600nm reaches 0.4-0.6.
4. Then make the below combinations into the 96 well plate for the plate reader to take readings.

	1	2	3	4	5	6	7	8	9	10	11	12
	No cells + 1mM	0.1mM	0.2m	0.3mM	0.4mM	0.5mM	0.6mM	0.7mM	0.8mM	0.9mM	1mM	Cell + No IPTG
LB(µL)	90	94	93	92	91	90	89	88	87	86	85	95
Cells(µL)	-	5	5	5	5	5	5	5	5	5	5	5
10X IPTG(µL)	10	1	2	3	4	5	6	7	8	9	10	-




5. Readings were taken, using a plate reader capable of shaking and heating to 37˚C, for BL21 as control, pT7-lysis cassette in BL21 plysS. (all performed in duplo).