Team:Queens Canada/Parts/Contributions

The PCR-Ligation Method of BioBrick Assembly allows the ligation of two linear BioBrick Parts surrounded by the BioBrick prefix and suffix, hereon referred to as Part A (red) and Part B (blue).They can be permanently ligated to each other using the SpeI cut site of Part A and the XbaI cut site of Part B. This linear ligation product is then PCR amplified using the Left Primer of Part A and the Right Primer of Part B. The PCR product then undergoes gel electrophoresis, and the DNA product corresponding to the right length is extracted and purified for further use.

During the ligation process, only one of the digestion enzymes, XbaI and SpeI is removed, and one remains in the ligation mixture. If the correct ligation occurred, a permanent scar will result from the ligation of XbaI sticky end to SpeI sticky end that is no longer recognized by either XbaI or SpeI digestion enzymes. The one enzyme remaining in the solution will serve as a selection mechanism for the correct ligation product, and prevent the re-ligation of digested fragments. The removal of one enzyme is necessary for the procedure. If both enzymes remain in the ligation mixture, undesired ligation can occur in which Part B will be ligated to Part A due to the double sticky ends created. However, if both digestion enzymes are removed, there will no longer be a selection mechanism for the correct ligation product.

The PCR Amplification of the ligation product using the left primer of Part A and the right primer of Part B serves as another selection mechanism for the correct ligation product, because only the correct ligation product will be amplified in the PCR reaction using those two primers. In addition, the PCR Amplification using high fidelity polymerase system improves the fidelity of this assembly method. Each step of the assembly only requires one PCR reaction for amplification instead of two bacterial incubation periods. This shortens the amplification time required for the assembly of a construct containing 3 BioBrick parts from 4 bacterial incubation periods to 2 PCR reactions.

The PCR-ligation method of BioBrick assembly is easy to debug once errors arise during the assembly. The PCR amplification product after each step of the assembly undergoes gel electrophoresis. Only the DNA product corresponding to the correct length of the desired ligation product is extracted and purified for further use. In order for the assembly to proceed to the next step, the previous step of the assembly must be successful. Since every step of the PCR-ligation assembly is checked before further processing, the number of potential errors that need to be accounted for during troubleshooting is significantly reduced compared to the convention methods of assembly.

Like the conventional methods of assembly, the linear product of PCR-ligation also contains the BioBrick prefix at its 5’ end and the BioBrick suffix at its 3’ end. Therefore, this method of assembly can theoretically be repeated an infinite number of times for the assembly of multiple BioBrick parts.

PCR Ligation, with one cleanup (above)

PCR Ligation, without cleanup (below)