Novel Assembly Method: PCR Ligation
While an effective method, our team felt the standardized method of BioBrick assembly had two key issues which limited it's effectiveness:
Undesired Ligation Products: Both the Standard BioBrick Assembly and the 3A Assembly require the ligation of the desired insert into one of the standard BioBrick plasmid vectors for amplification. During the digestion and ligation process, the plasmid bones and the desired inserts may ligate in many possible configurations and may generate undesired ligation products
Lengthy Ligation Time: The conventional method is very time- intensive. The Standard BioBrick Assembly Standard and the 3A Assembly allow only the assembly of two BioBrick parts at once. Consequently, the assembly of a construct containing 3 BioBrick parts, will require at least 4 incubation periods to complete assuming each step of the assembly is successful on the first attempt.
To improve the assembly progress, our team examined the possibility of using the polymerase-chain reaction (PCR) to complete ligations.
Mechanism
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)
Protocol
Materials
- Thermocycler
- PCR Tubes (0.2mL)
- KAPA HiFi™ Hotstart Kit
- 10 µl of each of the 2 linear BioBrick parts to be ligated
- 10 µM solution of left primer of BioBrick part A
- 10 µM solution of right primer of BioBrick part B
- Fermentas FastDigest SpeI system
- Fermnetas FastDigest XbaI system
- KAPA Fast Ligase system
- TBE (pH = 8.0)
- 3 µl of Ethidium Bromide
- Biotechnology grade Agarose
- Gel Box
- Power Supply
- Gel Supply
- Loading Dye
- Bio Basics EZ-10 PCR Product Purification Kit
- Transilluminator
Method
1. Digest linear BioBrick part A with the SpeI FastDigestion system, and digest linear BioBrick part B with the XbaI FastDigestion system.
2. Perform enzymatic cleanup for one of the digest mixtures using EZ-10 PCR Product Purification kit.
3. Mixing of the clean-up product and other digestion product in a 1:1 ratio to obtain a 10 µl mixture.
4. Use the 10 µl mixture and the Fast Ligase System to ligate the two BioBrick parts together.
5. Use the ligation product from the previous step as the template DNA for PCR amplification. Use the 10 µM solution of left primer of BioBrick part A and the 10 µM solution of right primer of BioBrick part B as primers for the PCR Amplification.
6. Follow the instructions in the KAPA HiFi™ Hotstart Kit to perform the PCR reaction. Only 20 to 25 thermocycles are necessary to amplify the ligation product.
7. Create a 1% agarose gel for electrophoresis of the PCR products.
8. With loading dye, load the entire digestion mixture to the 1% agarose gel. Perform gel electrophoresis at 100V and 400 mA for 60 minutes.
9. Using transilluminator, gel extract the visible DNA product corresponding to the right length of the ligation product.
10. Follow the instructions in the Bio Basics EZ-10 Purification Kit, perform purification of the gel extracted products.
11. This gel extracted product may be used for further assembly and processing.
Applications
This type of method is applicable DNA construct creation. A Request for Comments (RFC) document is currently being prepared.
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