Team:DTU-Denmark-2/Project/Other assembly systems

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Other assembly systems

Standerd BioBrick Assembly

https://2010.igem.org/Team:ESBS-Strasbourg/Results/Assembly -->new standard assemby http://ginkgobioworks.com/support/BioBrick_Assembly_Manual.pdf

Gibson Assembly

Gibson Assembly is an isothermal, single-reaction method for assembling multiple overlapping DNA molecules. The method was developed by Daniel G. Gibson at the J. Craig Venter Institute in 2009.
The assembly system employs 5´-T5 exonuclease, Phusion DNA polymerase, and Taq lig. Gibson can be used to assemble both ssDNA and dsDNA fragments. This methode makes it possible to join DNA molecules there are as large as 583kb and clone joined products in ''E. coli'' with a length up to 300kb. Among the advantages is that it takes the same amount of time to ligate n DNA fragments than two.

The way it's done

The isothermal One-step The 5´-T5 exonuclease removes the bases from the 5'-end of double strained DNA molecule, leaving a recess in the DNA. The ssDNA overhang is used to assemble the DNA fragments.
The T5 exonuclease are inactivated during the incubation at 50ºC. Phusion polymerase and Taq ligase fills the gaps of the annealed complementary ssDNA overhangs and seals the nicks in the end, leaving a joined DNA molecules.

Gateway assembly

MultiSite Gateway Assembly enables the assembly of multiple DNA fragments by utilizing site-specific recombination, and are provided by Invitrogen. The Gateway Assambly can clone up to 4 DNA fragments into one vector with by flanking the PCR products with specific att sites, and further directed recombination into the vector.

The way it's done

First PCR fragments with the appropriate att sites and orientation has to be constructed as shown in figure ?? As in the present version of Gateway, to enter MultiSite Gateway, sets of Entry Clones are obtained or generated. Entry Clones are mixed together with the appropriate MultiSite Gateway Destination -

In fusion

Sandwich

3A

https://2010.igem.org/Team:Bielefeld-Germany/Project/Protocols#3A_assembly

@@ Line 91: / Line 91: @@
 <a name="The way it's done"></a><h4><b>The way it's done</b></h3><br>
-The isothermal One-step The 5´-T5 exonuclease removes the bases from the 5'-end of double strained DNA molecule, leaving a recess in the DNA. The ssDNA overhang is used to assemble the DNA fragments.<br> The T5 exonuclease are inactivated during the incubation at 50C. Phusion polymerase and Taq ligase fills the gaps of the annealed complementary ssDNA overhangs and seals the nicks in the end, leaving a joined DNA molecules.
+The isothermal One-step The 5´-T5 exonuclease removes the bases from the 5'-end of double strained DNA molecule, leaving a recess in the DNA. The ssDNA overhang is used to assemble the DNA fragments.<br> The T5 exonuclease are inactivated during the incubation at 50ºC. Phusion polymerase and Taq ligase fills the gaps of the annealed complementary ssDNA overhangs and seals the nicks in the end, leaving a joined DNA molecules.
 <a name="Gateway Assembly"></a><h2><b>Gateway assembly</b></h2>
@@ Line 98: / Line 98: @@
 <br><br>
 <a name="The way it's done"></a><h4><b>The way it's done</b></h3><br>
-Restriction of
+First PCR fragments with the appropriate att sites and orientation has to be constructed as shown in <b>figure ??</b>
-  As in the present version of Gateway, to enter MultiSite Gateway, sets of Entry Clones are obtained or generated. Entry Clones are mixed together with the appropriate MultiSite Gateway Destination --> http://tools.invitrogen.com/Content/SFS/ProductNotes/F_Multisite%20Gateway%20Cloning-050310-RD-TL-HL0506021.pdf
+  As in the present version of Gateway, to enter MultiSite Gateway, sets of Entry Clones are obtained or generated. Entry Clones are mixed together with the appropriate MultiSite Gateway Destination -
 <br><br>