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

Comparison table

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 preferably be either 500 bp smaller or larger from each other as well as the backbone. The system is based on that one of the parts to be assembled is in the destination vector from the beginning. The assembly can be performed with 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 restriction sites of the four restriction enzymes; EcoRI and XbaI should be located upstream of the BioBrick part and SpeI and PstI downstream of the BioBrick part. 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 BioBricks also have to be without any of the four restriction sites. These restriction sites are used to cut the BioBrick to be inserted to obtain sticky ends and open the second receiving plasmid also obtaining sticky ends.

The resulting insert and open vector must be purified so unwanted and unspecified parts can be removed. The insert and cut plasmid are mixed and under the right conditions, making the sticky ends to form hybridization. Subsequently, a ligation is performed to re-ligated to form a stable DNA backbone plasmid, which can be transformed into E. coli cells. [1].

A major disadvantage 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.

3 antibiotic assembly

3A assembly is a method for assembling two BioBrick parts at time. 3A assembly relies on three way ligation (between the two parts and the backbone vector), thereby it differs from Standard Assembly, which use two way ligation between a part and a part + vector. The method 3A assembly was designed to make the gel purification of the digested parts unnecessary. Furthermore, antibiotic selection is used to eliminate unwanted background. The assembled parts can either be in two plasmids or generated by PCR.

The way it's done

The process of assembly with 3A resembles the Standard Assembly method. The 3A assembly exploits as wells the restriction sites of EcoRI, SpeI, Xbal and PstI to flank the BioBricks and destination vector.

 

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 in E. coli cells, the selection can be done by positive or negative selection [2].

The 3A method is still based on use of restriction enzymes digestion and ligation, which means the need for eliminating any illegal restriction as well as leaving a scar between the BioBricks. An other disadvantage with 3A assembly is the requirement for plasmids to contain three different antibiotic markers. Furthermore, the 3A 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 fragments. 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 method 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 number of DNA fragments as for two DNA fragments.

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].

One of the disadvantages by the Gibson assembly is that the primers for the assembly are more expensive due to the 40 bp extra nucleotides the primers have to be flanked with. The Gibson assembly is not as specific as the USER cloning, which cuts with a uracil instead of chewing back from the end point.

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].

The biggest difference between the two assembly systems are the speed. The Gateway assembly takes longer time to perform 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].

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.