Let's do something together!

Collaboration in science is one of the most important steps to success. You are very good in your field but still, some piece of a puzzle is missing in order to see the whole picture? It is not a problem at all if you are open and not afraid to ask for help or to offer yours. Meeting other people while working on a science project is also essential. Other people with their fresh mind might easily notice a reason why something is not working properly, suggest solutions or inspire with a new direction. So, let's collaborate!

Team Vilnius-Lithuania Collaboration

We were fortunate to enjoy a productive collaboration with Team Vilnius-Lithuania over the summer. We were able to contribute a mechanistic model of the PURE cell free system which is documented in detail on our modelling collaboration page. Team Vilnius-Lithuania worked with us on wet lab protocols for the characterisation of DNA degrading switch which are outlined here.

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. Edinburgh to Vilnius Delivery Contents

Tube Label	Contents
DL2524 Culture	Liquid culture of the strain DL2524 (LB with chloramphenicol 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 of 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 100 ml LB with 1 ml overnight culture.

• Incubate at 37 °C, 220rpm 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 x g, 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 µl and flash freeze on dry ice. Store at – 80 °C. Aliquot 100 µl 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 4 ul 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 45 °C water bath.

• Incubate on ice for 2 minutes.

• Incubate at 37 °C for 1 hour.

• Plate on agar supplemented with 25 µg/ml ampicillin and add to 37 °C 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 8 µl of pCol to 50 µl of DL2524 culture.

• Incubate on ice for 30 minutes.

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

• Incubate on ice for 2 minutes.

• Incubate at 37 °C for 1 hour.

• Plate on agar supplemented with 25 µg/ml Tetracycline and 25 µg/ml Ampicillin and add to the 37 °C incubator. Having grown overnight pick colonies once again and prepare 1.5 ml overnight culture in LB with 25 µg/ml Tetracycline 25 µg/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.

3.6 Validation

In order to observe the DNA degradation as a result of our switches activation we propose to run gel electrophoresis periodically over the 24hours after maxicell induction. We would expect to see a number of small DNA fragments from the maxicells degraded chromosome and bands corresponding to our 2 constructs pImm and pCol. After degradation switch activation we would expect to observe the bands corresponding to our plasmid constructs dissapear as Colicin E2 degrades maxicell DNA. Due to our need to visualise potentially very small DNA fragments we propose to use SYBR Green I Nucleic Acid Gel Stain or some similar high sensitivity strain.