The key difference between Golden Gate and Gibson Assembly is that Golden Gate relies on the presence of restriction sites within a particular sequence to be cloned, while Gibson Assembly does not rely on the presence of restriction sites within a particular sequence to be cloned.
Over the past decades, molecular scientists have developed various standardized procedures that allow the easier assembling of multiple DNA fragments into a single piece. Therefore, cloning methods such as Restriction Enzyme Ligation, Gateway Cloning, Gibson Assembly, Golden Gate Assembly, and TOPO Cloning are widely used by researchers in scientific experiments. Therefore, Golden Gate and Gibson Assembly are two molecular cloning methods that allow the assembly of multiple DNA fragments into a single piece.
CONTENTS
1. Overview and Key Difference
2. What is Golden Gate Assembly
3. What is Gibson Assembly
4. Similarities – Golden Gate and Gibson Assembly
5. Golden Gate vs Gibson Assembly in Tabular Form
6. Summary
What is Golden Gate Assembly?
Golden Gate is a molecular cloning method that facilitates the assembly of multiple DNA fragments into a single piece. This method relies on the presence of restriction sites within a particular sequence to be cloned. It originated in 1996. This technique uses Type IIS restriction enzymes and T4 DNA ligase. These enzymes cut DNA outside the recognition sites. They can create non-palindromic overhangs. Therefore, multiple fragments of DNA can be assembled using combinations of overhang sequences.
Figure 01: Golden Gate
Golden Gate Assembly Procedure
Golden Gate technique has main three steps in its procedure:
- creation of overhangs in the cloning vector,
- assembly of multiple DNA inserts by using fragment-specific sequences in overhangs, and
- ligation.
Golden Gate assembly uses circular cloning vectors (destination vector). Furthermore, in this method, the restriction site is eliminated from the ligated product in order to carry out digestion and ligation simultaneously. A typical thermal cycle protocol for Golden Gate oscillates between 37 °C and 16 °C because 37 °C is optimal for restriction enzymes and 16 °C is optimal for ligases.
What is Gibson Assembly?
Gibson Assembly is a molecular cloning method that allows the assembly of multiple DNA fragments into a single piece. Unlike the Golden Gate method, this method does not rely on the presence of restriction sites within a particular sequence to be cloned. It was found by Daniel G. Gibson of the J. Craig Venter Institute. This technique is a type of sequence and ligase independent cloning (SLIC) method.
Figure 02: Gibson Assembly
Gibson Assembly Procedure
Gibson Assembly needs DNA fragments containing 20-40 base pairs overlapping with adjacent DNA fragments. Overlaps are added via PCR. Then these DNA fragments are added to the Gibson maser mixture that has three enzymes. This procedure is carried out under isothermal conditions (50 °C for 1 hour) using three different enzymes: exonuclease, DNA polymerase and DNA ligase. The exonuclease chews back DNA from the 5’ end. Thus, it does not inhibit polymerase activity and allow the reaction to proceed in one single process. The resulting single-stranded regions on adjacent DNA fragments can be annealed due to overlap sequences. DNA polymerase fills up the gaps by adding nucleotides. Finally, the DNA ligase covalently joins the DNA of adjacent segments. Normally, Gibson Assembly uses linearized destination vectors. Moreover, this method can simultaneously combine up to 15 DNA fragments based on sequence similarity.
Similarities Between Golden Gate and Gibson Assembly
- Golden Gate and Gibson Assembly are two molecular cloning methods.
- Both methods can assemble multiple DNA fragments into a single piece.
- These methods use destination vectors.
- Both methods utilize thermal cyclers for the assembly of multiple DNA fragments.
- They are used in site directed mutagenesis.
Difference Between Golden Gate and Gibson Assembly
Golden Gate is a molecular cloning method used in the assembly of multiple DNA fragments into a single piece relying on the presence of restriction sites within a particular sequence to be cloned, while Gibson Assembly is a molecular cloning method used in the assembly of multiple DNA fragments into a single piece without relying on the presence of restriction sites within a particular sequence to be cloned. This is the key difference between Golden Gate and Gibson Assembly. Furthermore, Golden Gate method uses a circular destination vector, while Gibson Assembly method uses a linearized destinations vector.
The following infographic tabulates the difference between Golden Gate and Gibson Assembly.
Summary – Golden Gate vs Gibson Assembly
Molecular cloning is a method used to assemble recombinant DNA molecules and direct their replication within host organisms. Golden Gate and Gibson Assembly are two molecular cloning methods that allow the assembly of multiple DNA fragments into a single piece. The Golden Gate method relies on the presence of restriction sites within a particular sequence to be cloned, while Gibson Assembly does not rely on the presence of restriction sites within a particular sequence to be cloned. Thus, this is the key difference between Golden Gate and Gibson Assembly.
Reference:
1. “Golden Gate Assembly.” New England Biolabs: Reagents for the Life Sciences Industry.
Image Courtesy:
1. “Golden Gate Assembly” By Lee Yun Jie – Own work (CC BY-SA 4.0) via Commons Wikimedia
2. “Gibson assembly overview” By Tobias Vornholt – Own work (CC BY-SA 4.0) via Commons Wikimedia