Protocols

Ex vivo DNA assembly

Introduction

We developed a DNA assembly system purely based on parts homology, which only uses E. coli lysate to carry out the reaction. Our work builds on the previous research by (...) which was published as the SLiCE and Ex vivo assembly method. The concept is very similar to that of a Gibson method: the parts to be assembled contain an overlapping homology region, which allows homologous recombination to occur. While the Gibson assembly utilises an expensive piece of kit, containing a 3' to 5' exonuclease, a DNA polymerase to fill the gaps and a ligase to seal the nick. The Ex vivo, as we like to call it "E.G., or E. coli gratiae" only uses E. coli lysate to carry out this reaction. The lysate in fact does contain all the cellular machinery necessary to recognise a homology and to repair DNA. This process if facilitated when the lysate contains three lambda proteins, which can be easily expressed in the strains used to produce it. These are the same protein that allow Lambda Red Recombineering Knock-Outs, i.e. Gam, Exo and Beta, which respectively protect linear DNA from RecBCD nuclease activity, cleave DNA 3' to 5' and promote annealing of complementary single strands.

Protocol

Reaction conditions, improved "quick ex vivo", prefix suffix homology and homology length.

Standard SLiCE reaction

- 50–200 ng linear vector
- Appropriate amount of insert DNA in a 1 : 1 to 10 : 1 molar ratio of insert to vector,
- 1 ul 10X SLiCE buffer (500mMTris–HCl (pH 7.5 at 25?C), 100mM MgCl2, 10mM ATP, 10mM DTT)

50ul reaction Quick Reaction

- 25ul of 2X T4 ligase buffer (66mM Tris-HCL. 10mM MgCl2, 1mM Dithiothreitol, 1mM ATP, 7.5% Polyethylene glycol (PEG6000), pH 7.6 @ 25°C)

Results

pictures, table of colony count vs. lenght of reaction, table of colony count of lacZ vs. length of homology.