Let's do something together!

Collaboration in science is one of the most important steps to succeed. You are very good at your field but struggling in the other? It is not a problem at all when you are open and you are not afraid to ask for help or you can help someone. Meeting other people while working on a science project is also essential. Other people with their fresh mind might easily notice why something may go wrong, suggest new ideas or just share their opinion. So, let's collaborate!

Modelling Collabortation

DNA Degradation Switch Protocols

1. Introduction

Our DNA Degrading Switch requires the transformation of 2 plasmids prior to maxicell induction in order to function. Construct pImm contains a coding sequence for Dnase Colicin E2’s immunity protein Imm2 and an upstream recognition site for homing endonuclease I-SceI. Construct pCol contains a coding sequence for Dnase Colicin E2. By transforming pImm into DL2524 (maxicell progenitor strain) culturing successful transformants, transforming construct pCol and carrying out maxicell induction the DNA degrading switch becomes active. By exposing DL2524 to arabinose homing endonuclease I-SceI is induced, this degrades the chromosome of DL2524 and linearises pImm preventing further expression of Imm2. With its immunity protein no longer being expressed Colicin E2 should become active around 24hrs later degrading the DNA remaining within our Maxicell.

2. Delivery Contents

Tube Label	Contents
DL2524 Culture	Liquid culture of the strain DL2524 (LB with chloramphenacol and 0.5% glucose).
DL2524 STAB	DL2524 in nutrient agar with chloramphenicol and 0.5% glucose.
ISce-1 Imm2 1	pSB1C3 with ISce-1 site and Imm2 genes.
ISce-1 Imm2 8	pSB1C3 with ISce-1 site and Imm2 genes.
E2 2	Full Colicin E2 gene with BioBrick prefix and suffix
E2 4	Full Colicin E2 gene with BioBrick prefix and suffix
1st	gBlock of 1st half of Colicin E2 gene (with over lap for 2nd part)
2nd	gBlock of 2nd half og Colicin E2 gene

3. Protocols

3.1 Make DL2524 Competent

DL2524 needs to be made competent before it can be transformed. • Inoculate a single colony of DL2524 into 10 mL LB and culture overnight at 37 C, 200rpm. • Inoculate 100mL LB with 1 mL overnight culture. • Incubate at 37 C, 200rpm until OD₆₀₀ = 0.3-0.6 (approx. 2 hrs). • Transfer to 2 x 50 mL Falcon and leave on ice for 30 mins. • Centrifuge at 400 x g, 5 mins, 4 C. • Resuspend pellet gently in 25 mL ice cold 0.1 M CaCl₂ • Incubate on ice for 30 min. • Centrifuge at 4000 xg, 5 min, 4 C. • Resuspend pellet gently in 1.25 mLice cold CaCl₂/Glycerol solution (1.7 mL 0.1 M CaCl₂, 0.3 ml 100 % glycerol). • Aliquot 100 ul and flash freeze on dry ice. Store at – 80 C Aliquot 100ul and flash freeze on dry ice. Store at – 80 C.

3.2 Constructing pCol

The first step is to digest product from tube E2 2 to give BioBrick overhangs. By using the digestion protocol from http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones but substituting 4ul of linearized plasmid backbone this part can be obtained and will now to referenced as E2. Having obtained E2 this needs to be inserted into plasmid pSB1T3 from the iGEM distribution kit again using the digestion protocol and now the ligation protocol from http://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones with E2 as the fragment to be inserted.