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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 24 hrs 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

• 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 100 ul 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 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 here

3.3 Transforming pImm

pImm needs to be transformed into strain DL2524 allowing Imm2 to build up in cells before transformation of pCol and production of Dnase Colicin E2.

• Add 8ul of pImm to 50 uL of competent DL2524 culture.

• Incubate on ice for 30 minutes.

• Heat shock for 60 seconds in 45C water bath.

• Incubate on ice for 2 minutes.

• Incubate at 37 C for 1 hour.

• Plate on agar supplemented with 25ug/ml Ampicillin and add to 37C incubator.

3.4 Transforming pCol

Having allowed Immunity Protein Imm2 to build up in cells overnight it is now possible to transform pCol into cells which will have an immunity to dnase activity due to build up of Imm2. Firstly we need to pick colonies that have been transformed with pImm previously and add to LB prepared with 0.5% glucose. Incubate at 37 C for 1 hour. We can now follow the same transformation protocol as previously.

• Add 8ul of pCol to 50ul of DL2524 culture.

• Incubate on ice for 30 minutes.

• Heat shock for 60 seconds in 45C water bath.

• Incubate on ice for 2 minutes.

• Incubate at 37C for 1 hour.

• Plate on agar supplemented with 25ug/ml Tetracycline and 25ug/ml Ampicillin and add to the 37C incubator. Having grown overnight pick colonies once again and prepare 1.5 mL overnight culture in LB with 25 ug/mL Tetracycline 25ug/mL Ampicillin and 0.5% glucose in falcon tube shaking in 37 C water bath.

3.5 Maxicell Induction

Our maxicell progenitor strain has now been transformed with all the parts necessary for operation of our DNA degrading switch. Maxicell induction will activate the kill switch due to expression of homing endonuclease I-SceI linearizing pImm. To perform DL2524 maxicell induction:

• Measure OD₆₀₀ of overnight culture.

• Dilute culture at 0.025 OD₆₀₀ in LB with 0.2 % glucose.

• Grow for 80 minutes at 37 C.

• Dilute culture 10 times with 0.5 % arabinose and LB.

• Incubate at 37 C shaking gently.