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SPLiCE: Difference between revisions
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<h3>SLiCE <i>Ex vivo</i> DNA assembly</h3> | <h3>SLiCE <i>Ex vivo</i> DNA assembly</h3> | ||
<h4>Introduction</h4> | |||
[[File:Lysate_illustration.png|thumbnail]] | [[File:Lysate_illustration.png|thumbnail]] | ||
We developed a DNA assembly system purely based on parts homology, which only uses <i>E. coli </i> lysate to carry out the reaction. Our work builds on the previous research on lysate based assembly methods, in the particular <a href="http://nar.oxfordjournals.org/content/40/8/e55.long">SLiCE</a> and <a href="http://journal.frontiersin.org/article/10.3389/fbioe.2013.00012/abstract"><i>Ex vivo</i></a>. 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 <i>Ex vivo</i>, as we like to call it "E.G., or <i>E. coli </i> gratiae" only uses <i>E. coli lysate </i> 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. In addition to normal lysate, this system was tested using a lysate of cells expressing lamda proteins. These are the same protein that allow Lambda Red Recombineering Knock-Outs, <i> i.e. </i> 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. | We developed a DNA assembly system purely based on parts homology, which only uses <i>E. coli </i> lysate to carry out the reaction. Our work builds on the previous research on lysate based assembly methods, in the particular <a href="http://nar.oxfordjournals.org/content/40/8/e55.long">SLiCE</a> and <a href="http://journal.frontiersin.org/article/10.3389/fbioe.2013.00012/abstract"><i>Ex vivo</i></a>. 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 <i>Ex vivo</i>, as we like to call it "E.G., or <i>E. coli </i> gratiae" only uses <i>E. coli lysate </i> 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. In addition to normal lysate, this system was tested using a lysate of cells expressing lamda proteins. These are the same protein that allow Lambda Red Recombineering Knock-Outs, <i> i.e. </i> 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. | ||
We decided to test the efficacy of SLiCE for the assembly of parts only based on a short flanking homology. This homology is roughly equivalent to that of the biobrick prefix and suffix. This means that a two part assembly, of the insert - such as a gblock - into the standard pSB1C3 vector would only require one Seamless Ligation step. This avoids the standard (and costly!) digestion/ligation steps that usually are required for the biobrick assembly. | We decided to test the efficacy of SLiCE for the assembly of parts only based on a short flanking homology. This homology is roughly equivalent to that of the biobrick prefix and suffix. This means that a two part assembly, of the insert - such as a gblock - into the standard pSB1C3 vector would only require one Seamless Ligation step. This avoids the standard (and costly!) digestion/ligation steps that usually are required for the biobrick assembly. | ||
We tested this approach by ligating the standard J04450 RFP generator to pSB1C3 using 22bp and 21bp of homology (biobrick prefix and suffix :) ) and successfully achieved pink, ligated colonies. | We tested this approach by ligating the standard J04450 RFP generator to pSB1C3 using 22bp and 21bp of homology (biobrick prefix and suffix :) ) and successfully achieved pink, ligated colonies. | ||
<h4>Materials and methods</h4> | <h4>Materials and methods</h4> | ||
<p> <br> | <p> <br> | ||
