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

From 2011.igem.org

(Difference between revisions)

Revision as of 20:33, 11 September 2011

Other assembly systems

Standard BioBrick Assembly

the Standard Assembly of BioBricks was first suggested by Tom Knight, and has subsequently been modified by other scientist to overcome hurdles in the Standard Assembly. In the Standard assembly their can only be assembled two parts in each cycle, making it time consuming to assemble more than two parts.

The way it's done

The Standard assembly uses the restrictionsites of four restriction enzymes; EcoRI and XbaI on the upstream and SpeI and Pst downstream of the BioBrick part on the vector. This sites are used to cut a DNA fragment (BioBrick) out of the plasmid and open the second and receiving plasmid. The resulting insert and open vector must be purified so unwanted and unspecified parts can be removed before the recombination of the insert and the cut plasmid.
The inset and cut plasmid are mixed and under the right conditions the sticky ends from the specified restriction will re-ligate, resulting in a vector containing both parts. The vector can now be transformed into E-coli.

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.Recombination is a efficient and quick way to assemble biobricks and are widely used. Invitrogen have designed standalized and simplified the technique in their Gateway Cloning.
Gateway Assembly uses two different bacteriophage recombination enzymes to assemble tje destination vector with the Entry clones. The process is extremely robust and furthermore overcome the steps by traditional restriction cloning.

The way it's done

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 idea with Gateway assembly is that it is executed in the same way whether you join two or four DNA fragments. Depending on the number of fragments specific att sites are attached, always starting with attB1 and ending with attB2. The att sites only differ in a few bases.
First PCR fragments with the appropriate att sites and orientation has to be constructed as shown in figure ?? and then assembled with a Entry Clone, containing the respective att sites. The Entry Clones are mixed together with the appropriate destination vector by a LR clonase reaction. The resulting expression clone is then ready for tranformation and functional assays.

In fusion

In-fusion BioBrick assembly are a methode for assembling of two or many Biobricks, provided by Clontech. The assembly system can be semi-standarlized by simple primer design rules, minimizing the time used on planning the assembly reactions.

The way it's done

The PCR fragments are assembled with the use of at least 15pb homology on both ends. The forward primer on the first PCR fragments must have homologes to the reverse primer of the second PCR fragment and so forth on every part of the BioBricks. Afterwards the PCR fragments can be fused into a pre-engineered vector by creation of single-stranded regions made by the In-fusion enzyme. The reaction is fast an efficient.

Sandwich

3A

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

@@ Line 79: / Line 79: @@
 <a name="Standard BioBrick Assembly"></a><h2><b>Standard BioBrick Assembly</b></h2>
 <br>
-the Standard Assembly of BioBricks was first suggested by Tomas Knight, and has subsequently been modified by other scientist to overcome hurdles in the Standard Assembly. In the Standard assembly their can only be assembled two parts in each cycle, making it time consuming to assemble more than two parts.
+the Standard Assembly of BioBricks was first suggested by Tom Knight, and has subsequently been modified by other scientist to overcome hurdles in the Standard Assembly. In the Standard assembly their can only be assembled two parts in each cycle, making it time consuming to assemble more than two parts.
 <br>
-<a name="Gateway Assembly"></a><h2><b>Gateway assembly</b></h2>
+<a name="The way it's done"></a><h4><b>The way it's done</b></h3><br>
 <br>
-The Standard assembly uses the restrictionsites of four restriction enzymes; EcoRI and XbaI on the upstream and SpeI and Pst on the right of the BioBrick part on the vector. The resulting insert and open vector must be purified before recombinated.
+The Standard assembly uses the restrictionsites of four restriction enzymes; EcoRI and XbaI on the upstream and SpeI and Pst downstream of the BioBrick part on the vector. This sites are used to cut a DNA fragment (BioBrick) out of the plasmid and open the second and receiving plasmid. The resulting insert and open vector must be purified so unwanted and unspecified parts can be removed before the recombination of the insert and the cut plasmid. <br>The inset and cut plasmid are mixed and under the right conditions the sticky ends from the specified restriction will re-ligate, resulting in a vector containing both parts. The vector can now be transformed into E-coli.
 <br><br>