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Circuit Construction with Diverse Methods

We developed a bacteria platform capable of efficient and theoretically-infinite gene insertion. As to demonstrate our system feasibility and functionality, we designed 11 different circuits respectively located on chromosome and plasmids to cooperate and implementing the brilliant function of “super insertion”.

Although many of these DNA elements constituting circuits can be found in the iGEM official registry, DNA element assembly remains a big challenge. In spite of iGEM official recommending assembly method, 3A assembly, circuit construction still seems quite inconvenient and time-consuming, because 3A assembly needs a lot of time to proliferate the ligation fragments into cells and less preferably leaves a scar between two ligation fragments.

Figure 1 - Multiple fragment assembly with seamless assembly method. These method depends on bacteria proliferation. Firstly, add two kinds of homology sequences to fragments; that is, add 15~20bp plasmid homology sequences to fragment A and C, and add 9bp linear DNA homology sequences to each fragments. Construct a plasmid, transform, proliferate and serve as a template to amplify the assembly fragment (A+B+C).

Figure 2 - schematic illustration of the protocol of seamless cloning. Add two homology sites (15~20bp) of the linear plasmid backbone to each ends of any fragments by PCR. Then the fragment and the plasmid can form a circular DNA without scar.

Figure 3 - Two linear fragment assembly mediated by seamless cloning. Two rounds of PCR are needed. The first round aim at introduce mutual homology sites of both fragments: 3’ end of fragment A with the 9bp 5’ end of fragment B; 5’ end of fragment B with 9bp 3’ end of fragment A. The second round of PCR followed by incubating into seamless cloning reaction system and lead to a large number of assembly fragment (A+B).

In consideration of efficiency and convenience, we mainly use an no-scar assembly methods. Different from the famous assembly method, Gibson assembly, our method for assembling the whole circuit is no-scar recombination, also known as seamless cloning or seamless assembly. The method do not depend on restriction enzyme digestion and ligase activity, and thus is not restricted to DNA fragment restriction enzyme site introduction. Based on homologous recombination, certain recombinases function in the in vitro system. What needs to do is add certain length homologous sequences of the second fragment’s end to the first ones. Then the recombinase recognizes the homology site and links all the modified fragments together.

The seamless cloning or assembly method is quite an efficient way to construct a complex circuit. Different from Gibson assembly, the assembly method relies on plasmid backbone and bacteria cloning. Exactly speaking, the recombinase-based assembly method, in fact, is divided into two kinds, each of which consists of different components. The first one is called seamless cloning which is only applicable for inserting fragments to linear plasmid backbone. As Fig. 1 shows, seamless cloning requests adding two homology sequences (15-20bp) of plasmid backbone to each ends of the inserting fragment.

Figure 4 - PCR product after applying seamless cloning to two fragments (60bp, 900bp). PCR with each fragment primer and obtain the combining fragments (960bp).

Figure 5. Multiple fragment assembly PCR results. Three different fragments are involved with respective length (1300bp, 60bp, 1500bp).

Figure 6. Two fragment assembly using over-lap PCR. The assembly fragment of 2100bp is combined with 1200bp and 900bp fragment.

After optimizing the reaction system, we developed a new protocol to use the assembly method in a different way. That is, we use seamless cloning assembly to ligase two linear fragments by following PCR. As an example, after adding 9bp of fragment B (60bp) 5’ end to 3’ end of fragment A (900bp) by PCR, adding 9bp of the 3’ end of fragment A to 5’ end of fragment B in the same way, and incubating in the seamless cloning reaction system, fragment A + B can be get by PCR using 5’end primer of A and 3’ end primer of B. As the figure shows, fragment A+B (960 bp) gets successfully proliferated.

When confronting multiple fragment assembly, we use seamless assembly to integrate these fragments to a plasmid backbone. Proliferate the plasmid in bacteria cells and use it as a template to amplify the targeting sequence by PCR. As an example, after PCR modification of three fragments ( A,1300bp,B,60bp,C,1.5bp ) and incubation in seamless assembly reaction liquid, these three fragments can be inserted into the plasmid in a designed direction (A、B、C). Transform, proliferate bacteria cells, PCR and then we get the assembly fragment (about 2.9kb).

Additional approach is over-lap PCR. In situation where assembly fragment cannot be got via PCR due to some unknown reasons, we would like to select over-lap PCR method. For instance, Frag A (900bp) and Frag B (1200bp) are two fragments which needs assembling together. After applying to over-lap PCR, the linking fragments (about 2100bp) are amplified and located on specific position through gel electrophoresis.

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