Team:Jilin China/Construction

CONSTRUCTION


Enzyme Golden Gate Intermediate

Construction

  • Type IIS Restriction Endonuclease

    Restriction enzymes are traditionally classified into four types on the basis of subunit composition, cleavage position, sequence specificity and cofactor requirements[1]. Our commonly used restriction endonucleases, such as EcoRI, belong to type II restriction endonucleases, which recognize specific 4 to 8 base-pairs. The recognized sequence is inverted repeats and the cleavage site is located in the recognition site. After enzymatic cleavage and the sticky end or blunt end is produced. The cleavage diagram is as follows:

    R II

    R IIS

    R IIs

    Type IIS restriction enzymes differ from other Type II restriction enzymes in several ways.These enzymes recognize sequences comprise two distinct sites, one for DNA binding, the other for DNA cleavage. Type IIS restriction endonucleases cleave DNA at a defined distance from their non-palindromic asymmetric recognition sites, creating 4-base overhangs.

    Type IIS cleavage sites have no inherent sequence-specificity, so the sequence of the overhang they generate varies from one recognition site to another. Fragments produced by Type IIS-digestion of natural DNA molecules generally have different overhangs, therefore, and will not anneal to one another.

    However, if the sequence of the overhang is predetermined, by designing it into a PCR primer, for example, then it can be made to complement another and to be directional. This feature is used to great advantage in 'Golden Gate' assembly where multiple fragments can be stitched together in the correct order and orientation in a single ligation. The advantage of using Type IIS enzymes for assembly is that the recognition sequence can be placed in the primer on either side of cleavage site. If placed inside, 3' to the cleaved end, it will be retained in the construct and can be re-used subsequently. If placed outside, 5' to the cleaved end, it will be lost, leading to a ‘scar-less’ assembly. This characteristic is widely used to perform in-vitro cloning techniques such as Golden Gate cloning[2].

  • Golden Gate Assembly

    Golden Gate Assembly is a one-tube efficient cloning method based on Type IIS restriction enzymes that cleave outside their recognition sites and leave 4-base overhangs[3]. By adding the recognition site of type IIS enzyme on the plasmid, it will be digested by enzyme and will not appear at the end constructs. The fragment gene of interest contains complementary sticky ends can ultimately be assembled by ligation[4].

    Commonly used IIS type restriction enzymes are as follows:

    Enzyme Recognition Sequence
    BbsI GAAGAC (2/6)
    BsaI GGTCTC (1/5)
    BtgZI CGTCTC (1/5)
    BsmBI GCGATG (10/14)
    Esp3I CGTCTC (1/5)
    SapI GCTCTTC (1/4)

    We chose BbsI and BsaI type IIS restriction enzymes this year.

    The procedure of construction is annealed DNA oligos, phosphorylation and Golden Gate assembly:

    1. Annealed oligos

    Oligos annealing can be use to add any short stretch of DNA to a plasmid. Most of the thermosensors we designed are 40~80bp, so we can design a forward oligo with the sequence of thermosensor and with the reverse oligo being the reverse compliment so that they could anneal. We also need to make sure our annealing products contain the cohesive end bases to complement the overhangs on the plasmid. The cohesive end bases could be a part of thermosensor, because we use the Golden Gate assembly, which has no-scar products.

    Figure 1. Diagram of Annealed oligos

    2. Phosphorylation

    After annealing, we do the 5' phosphorylation of DNA for the subsequent ligation. Because all ligations require a 5' phosphate, which is generated through restriction dgestions or kinase treatment of fragments. Oligos ordered from a supplier would not come with a 5' phosphate unless you specify that modification is necessary. If oligos were not phosphorylated, we need to phosphorylate fragments with T4 Polynucleotide kinase prior to ligation. The T4 polynucleotide kinase can catalyze the transfer and exchange of P from the γ position of ATP to the 5'-hydroxyl terminus of polynucleotides and 3'-monophosphates.

    Figure 2. Diagram of Phosphorylation

    3.Golden Gate assembly

    We use Type IIS restriction enzyme to digest the plasmid and use T4 DNA ligase to assemble DNA fragments we have phosphorylated before.

    Figure 3. Diagram of Golden Gate assembly

    We have recorded the detailed protocol of the Golden Gate assembly on the protocol page. Other users can refer to our protocol to design their own experiments (the amount of each added reagent in the protocol has been reduced to a minimum, it is not recommended to continue to reduce the amount. At the same time, when selecting other types of Type IIS restriction endonucleases, you should test whether the enzyme can work normally in T4 buffer. We have tested BsaI and BbsI with T4 buffer from NEB and Takara, which works well. But others hasn't been tested yet.) .

  • Construction Intermediate

    Since we need to build more than 400 components, if we use the traditional assembly method, it will be a huge amount of work. Due to the low efficiency and success rate of traditional assembly, which will seriously delay our experimental process. We finally decide to use Golden Gate Assembly after discussion. However, the selection of the correct construct is also a problem, hence we designed the following Construction Intermediate to select the correct constructs by visible color changes of the colonies.

    Figure 4. Diagram of Construction procedure

    The Construction Intermediate includes a promoter J23104, a pigment protein tsPurple (BBa_K1033906), a pigment protein cjBlue (BBa_K592011), a BbsI site, and a BsaI site. Our Construction Intermediate has three stages of Open Reading Frame (ORF). Promoter (J23104)+cjblue in Part I can be replaced with other promoter sequences by using BbsI restriction enzymes, and the tsPurple in Part II can be replaced with RNA thermosensor sequences by using BsaI restriction endonuclease. At the first stage of OFR, the colonies are in blue due to the expression of cjBlue. After the first replacement of the promotor, the colonies in Stage B are in purple. After the second replacement, the colonies with the correct constructs show their own milky white. Color changes can refer to the following table:

    We can successfully construct more than 200 plasmids in one day. And the assembly success rate is over 95%, which is a very exciting result.

  • References

    • [1]NEB. Types of Restriction Endonucleases[EB/OL]. https://international.neb.com/products/restriction-endonucleases/restriction-endonucleases/types-of-restriction-endonucleases
    • [2]NEB. Everything You Ever Wanted to Know About Type II Restriction Enzymes[EB/OL]. https://international.neb.com/tools-and-resources/feature-articles/everything-you-ever-wanted-to-know-about-type-ii-restriction-enzymes
    • [3]NEB. FAQ: Which restriction enzymes are used in Golden Gate Assembly?[EB/OL]. https://international.neb.com/faqs/2017/07/17/which-restriction-enzymes-are-used-in-golden-gate-assembly
    • [4]Engler C, Kandzia R, Marillonnet S. A one pot, one step, precision cloning method with high throughput capability[J]. Plos One, 2008, 3(11):e3647.