Difference between revisions of "Part:BBa K1433013:Experience"

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Applications of BBa_K1433013

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.

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 (图1). As Fig. 1 shows, seamless cloning requests adding two homology sequences (15-20bp) of plasmid backbone to each ends of the inserting 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.

Figure 1 - PCR product after applying seamless cloning to two fragments (60bp, 900bp). PCR with each fragment primer and obtain the combining fragments (960bp).

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 - 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.

User Reviews

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 <p>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.</p>
 {| align='center'
-|[[Image:circuit construct1.png|530px|thumb|center|'''Figure 1''' - PCR product after applying seamless cloning to two fragments (60bp, 900bp). PCR with each fragment primer and obtain the combining fragments (960bp). ]]||[[Image:circuit construct2.png|1200px|thumb|center|'''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.]]
+|[[Image:circuit construct1.png|530px|thumb|center|'''Figure 1''' - PCR product after applying seamless cloning to two fragments (60bp, 900bp). PCR with each fragment primer and obtain the combining fragments (960bp). ]]||[[Image:circuit construct2.png|1200px|thumb|center|'''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.]]||[[Image:circuit construct3.png|1200px|thumb|center|'''Figure 3''' - 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.]]
 |}