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

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	1 Standard BioBrick Assembly 2 3A 3 Gateway system 4 Gibson Assembly 5 In fusion 6 Comparison	Standard BioBrick Assembly The Standard Assembly of BioBricks was first developed by Tom Knight, and has subsequently been modified by other scientist to overcome hurdles in the Standard Assembly. Only two parts larger than 200 bp can be assembled by the Standard assembly in each cycle. Furthermore, the parts should preferely be either 500 bp smaller or larger from each other as well as the backbone. The assembly system depends on two ligases, meaning that the one assembles part has to be in the destination vector from the beginning. The assembly can then be performed either suffix insertion (insertion of the added part behind the existing part), or a prefix insertion (insertion of the added part in front of the existing part). 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. For adequate results it is necessary to ensure a plasmid with only the given four restriction sites, if other restriction sites are present, they have to be eliminated by alteration. Furthermore, the parts or BioBrick wish This sites are used to cut the DNA fragment the plasmid and open the second and receiving plasmid. For adequate results it is necessary to ensure a plasmid with only the given four restriction sites, if other restriction sites are present, they have to be eliminated by alteration.   The resulting insert and open vector must be purified so unwanted and unspecified parts can be removed. after purification on a gel, the insert an the cut plasmid are assembled by recombination. The inset and cut plasmid are mixed and under the right conditions, making the sticky ends from the specified restriction to re-ligate, resulting in a vector containing both parts. The vector can now be transformed into E. coli [1]. Difference between Plug'n Play assembly and Standard Assembly The biggest disadvantages for the Standard assembly is the need for restriction digestions, ligations and the need for site-directed mutagenesis if more restriction sites are available on the plasmid. The limitation in only assembling two parts at the time, makes the Standard assembly much more time consuming. And last the scars made by the assembling, make it impossible to create fusion proteins by the Standard assembly. 3A The 3A assembly, like the Standard assembly, only join 2 BioBrick parts. To select the correct assemblies, the method uses the function of antibiotics which minimize time compared to gel purification. The way it's done The assembled parts can either be in two plasmids or generated by PCR. The Methode uses the restriction sites of EcoRI, SpeI, Xbal and PstI to flank the biobricks and destination vector. The difference with 3A and standard assembly is also that 3A uses three way ligation instead of only two.   Digestion of the two parts and the destination plasmid are done so sticky ends are compatible with the wanted assembly of the vector. The restriction digest and ligation has to be executed in two separate steps. After transformation into competent E.coli cells, the selection can be done by positive or negative selection.[2] Difference between Plug'n Play assembly and 3A assembly One of the disadvantages with 3A assembly is the need for plasmids to contain three different antiobiotic cassettes, beside the use of restriction digest and ligation leaving a scar on the plasmid. This method is also only capable in combining two parts at a time. 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, ready for transformation[3]. Difference between Plug'n Play assembly and Gibson Assembly One of the disadvantages by the Gibson assembly is that the primers for the assembly is more expensive. The Gibson assembly is not as specific as the USER cloning, which cuts with a urasil instead of chewing back from the en. 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 the figure below.   The att flanked PCR can then be 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 [4]. Difference between Plug'n Play assembly and Gateway assembly The far most difference are the speed with which the assembly takes place. The Gateway assembly take a longer time and are more complex than the Plug'n Play. 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. The assembly system can be seen below, and can work with either the upstreame or downstreme PCR amplification of the vector +gene.   Afterwards the PCR fragments can be fused into a pre-engineered vector, containing a antibiotic resistance gene, by creation of single-stranded regions made by the In-fusion enzyme. [5]. Difference between Plug'n Play and In-Fusion assembly The In-fusion reaction is fast an efficient but one of the disadvantages is that the suppliers are mote expensive, costume primers and occasionally mutations occur in the products making the no good transformation. Comparison of the different assemblies Assembly system Speed Scars Res. digestion Multiple part assembly Standard BioBrick Slow √ √ No, two 3 A Slow √ √ No, two Gibson Medium-Fast No No Yes Gateway Slow-Medium No No Yes In fusion Fast No No Yes Plug'n Play Fast No No Yes