Speaking of molecular cloning, almost every partner who has done plasmid construction knows Gibson assembly. This method is so popular! I remember when I first entered the laboratory, my brothers and sisters taught me that the method of constructing plasmid was Gibson assembly. At that time, everyone in the laboratory was assembling with Gibson, and I realized that the classic enzyme digestion and connection method learned in high school biology class and undergraduate biology class was long out of date. Later, I read a lot of literature and found that besides Gibson assembly, there are actually many useful plasmid construction methods, and Golden Gate assembly, which appeared almost at the same time as Gibson assembly, is one of them. Both methods can be used for seamless cloning.
Kinmen assembly is a new method of restriction enzyme ligation [1 2], which is different from the traditional method of restriction enzyme ligation. The traditional restriction endonuclease ligation method uses the standard type II restriction endonuclease (restriction endonuclease) (such as Eco RI) to cut DNA. These restriction enzymes usually recognize palindromes of 4~8 bp, and cut within the recognized sequence to produce sticky ends or blunt ends (Figure 1A). Golden Gate Assembly uses IIS restriction enzymes (such as Bsa I) to cut DNA. These restriction enzymes recognize non-palindromic sequences and cut them outside the recognized sequences to produce sticky ends. (fig. 1B).
Fig. 1II restriction endonuclease and IIS restriction endonuclease cut DNA.
The sticky end produced by IIS restriction endonuclease cutting DNA is usually 2 or 4 bases. In the ligation reaction, the more bases at the sticky end, the higher the fidelity of the ligation product. Therefore, Golden Gate assembly usually uses IIS restriction endonucleases that can produce sticky ends of 4 bases, such as Bsa I, Bsm BI and Bbs I (Figure 2). NEB company carried out molecular modification on these three wild-type restriction endonucleases, and obtained three restriction endonucleases with the same recognition sequence but stronger specificity, namely Bsa I-HFv2, Bsm BI-v2 and Bbs I-HF (Figure 2). The recognition sequence length of these restriction enzymes is 6 bp, so the probability of their appearance in DNA sequence is 1/4096.
Fig. 2 Specificity of three IIS restriction endonucleases
As shown in fig. 2, the specificities of three types of IIS restriction endonucleases can be recorded as BSA I (GGTT TCTC1/5), BSMB I (CGTT TCTC1/5) and BBS I (GAAAAC 2/6) respectively. In addition, IIS-type restriction endonuclease SAP I (GCT CTTC1/4), which can produce 3-base sticky end, is often used [3]. Although the sticky end produced by this restriction endonuclease is short, the fidelity of the ligation product will be slightly reduced, but the recognition sequence is long (7 bp), so the probability of its appearance in DNA sequence is 1/65438+. According to the sequence of cleavage sites, Bsa I, Bsm BI and Bbs I can produce 256 kinds of sticky ends, and Sap I can produce 64 kinds of sticky ends.
There are many cloning kits based on Kinmen assembly in the market, but the prices are very expensive. I always suggest that you buy your own enzymes and reagents to prepare the reaction system. As long as you know the working principle and experimental design method of Kinmen module, there is no problem. Next, I will take Bsa I-HFv2 as an example to talk about how to use Kinmen to assemble and construct plasmids.
Design primer
The recognition sequence of Bsa I was introduced by PCR and added to the 5' end of the primer. In order to ensure that the restriction endonuclease can stably combine with DNA double strand and play a cutting role, a protective base should be added at the end of the recognition sequence. The number and type of protecting groups are not fixed. In order to simplify the primer design, we can use TTT as the protective base (3 bp is enough for most restriction enzymes, and the protective base can be increased to 6 bp for insurance. Because the cleavage site is downstream of the recognition sequence and can be any sequence, there are three different primer design methods, as shown in Figure 3.
Fig. 3 Three strategies for designing primers: N represents any base, 1234 and 5678 represent two different cleavage sites, and N and cleavage sites can be introduced by using the existing sequence of PCR template or primer.
Experimental principle
Connection of (1) 2 DNA fragments
Fig. 4 The golden gate assembly connects two segments.
(2) the connection of four DNA fragments
Fig. 5 The Golden Gate Assembly connects four fragments:1234,5678, and ABCD and efgh represent four different cleavage sites.
The above two pictures show the working principles of 2-segment connection and 4-segment connection respectively, and the connections of other segments are similar. Enzymatic digestion and ligation are carried out in the same reaction system, and PCR products can be used as substrates (as shown in Figures 4 and 5), or PCR products can be cloned into plasmid vectors first, and then plasmids can be used as substrates. Under suitable experimental conditions and design, more than 20 DNA fragments can be assembled by using Kinmen assembly. The following video can help you further understand the working principle of Golden Gate assembly.
Working principle of golden gate module
operating procedure
(1) preparation reaction system
① The addition amount of each insert can be converted according to the addition amount of the carrier and the length of the carrier and the insert. Here is a conversion tool/#! /ligation; ? ② other available endonucleases: BsmBI-v2(NEB #R0739, 10U/? l)、BbsI-HF (NEB #3539， 10U/？ L), Sapi (NEB #R0569, 10U/? l)； ③ If the number of inserted fragments is 1~ 10, use 10 units; ; If the number of inserted fragments is greater than 10, 20 units are used; ④ If the number of inserted fragments is 1~ 10, 500 units will be used; ; If the number of inserted fragments is greater than 10, 1000 units are used.
(2) reaction procedure
Note: 37℃ is the optimum reaction temperature of BsaI-HFv2. If other restriction enzymes are used, this temperature may need to be adjusted. For example, the optimum temperature of BsmBI-v2 is 42℃.
superiority
Compared with the traditional restriction endonuclease ligation
The cleavage site of (1) IIS restriction endonuclease is outside the recognition sequence, so the recognition sequence is removed during the restriction enzyme digestion, and unnecessary sequences will not be introduced into the ligation product.
(2) IIS restriction endonuclease has no requirement on the sequence of the cleavage site, so different sticky ends can be generated by designing the sequence of the cleavage site, which makes it possible to directionally assemble multiple fragments.
(3) Because the recognition sequence will not remain in the ligation product, the digestion reaction and ligation reaction can be carried out in the same reaction system, which simplifies the experimental process.
Compared with Gibson assembly
The ligation process of (1) fragment does not involve sequence degradation and synthesis, so the fidelity of the ligation product is higher.
(2) More DNA fragments can be assembled, which is suitable for plasmid libraries.
(3)? DNA fragments with homologous sequences or repetitive sequences can be assembled, for example, it can be used to construct plasmids expressing multiple sgRNA [4].
(4)? Standardized synthetic biological elements can be constructed, similar to bio-bricks.
Matters needing attention
(1) A suitable restriction endonuclease should be selected, and the recognition sequence of this restriction endonuclease cannot appear in the final ligation product.
(2) When designing primers, the direction of recognition sequence and cleavage site should be correct.
(3) When the number of inserted fragments is large, in order to ensure the specificity of ligation, it is necessary to design the 4 bp sequence of cleavage site reasonably.
Table S7 in reference [5] provides the optimized sequence combination of cleavage sites.
References:
[1] engler et al. (2008)? PLoS One？ 3，e3647。
One-pot one-step accurate cloning method with Capabilitydx.doi.org of Qualcomm quantity
[2] engler, C. et al. (2009)? PLoS One？ 4，e5553。
Kinmen Reorganization: A One-pot DNA Reorganization Method Based on IIs Restricted Enzymesdx.doi.org
[3] Whitman, L. et al. (20 13)? Ginette. English Biotechnology. ? 33, 42.
(PDF) Cloning gene fragments quickly and without scar using System.www.researchgate.net Electra vector.
[4] Vad-Nielsen, J. et al. (20 16)? Cell molecules. Life science. ? 73, 43 15–4325.
https://doi . org/ 10. 1007/s 000 18-0 16-227 1-5 doi . org
[5] HamediRad, M. et al. (20 19)? ACS synthesizer. biology ? 8, 1047– 1054.
Efficient single pot without scar Jinmen Assemblydoi.org