Team:Jilin China/Construction

CONSTRUCTION


Construction

  • Type IIs Restriction Endonuclease

    Restriction enzymes can be divided into four categories according to their different enzyme digestion methods, named Type I, Type II, Type III and Type IV. Our commonly used restriction endonucleases, such as EcoRI, belong to type II restriction endonucleases, which recognize specific 4 to 8 nucleotide sequences . The recognized sequence is inverted repeats and the cleavage site is located in the recognition site. After enzymatic cleavage and the cleavage end or blunt end is produced. The cutting diagram is as follows:

    R II

    R IIs

    R IIs

    However, compared with the common Type II restriction enzymes the Type IIS restriction endonuclease is a relatively specific enzyme that cleaves DNA downstream of the recognition site, from the three-dimensional structure analysis of the enzyme. This is due to the fact that the recognition and catalytic regions of the Type IIS restriction enzyme are separated by a polypeptide linker.

    The advantage of the TypeII S restriction enzyme is that it has no requirement to a cleavage sequence. The cutting position can be any sequence, and multiple DNA fragments can be assembled through complementary ends. This assembly method is also called GoldenGate Assembly.

  • Goldengate Assembly

    The goldengate assembly relies on the type IIS restriction enzyme. By adding the recognition site of type IIS enzyme on the plasmid, it will be digest 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.

    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 goldengate assembly:

    1. Annealed DNA 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 top oligo with the sequence of thermosensor and with the bottom oligo being the reverse compliment so that they anneal. We also need to make sure to include the cohesive end bases to complement the overhangs generated when digesting the plasmid. The cohesive end bases can be a part of thermosensor, because we use the goldengate assembly, which is a no-scar assembly.

    2. Phosphorylation

    After annealing, we do the 5' phosphorylation of DNA for subsequent ligation. The T4 polynucleotide kinase can catalyze the transfer and exchange of P from the γ position fo ATP to the 5'-hydroxyl terminus of polynucleotides and 3'-monophosphates.

    3.Goldengate assembly

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

    We have recorded the detailed protocol of the goldengate 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 T4 ligase and T4 buffer from NEB and Takara, others did not tested yet.) .

  • Construction Device

    Since we need to build more than 400 components, if we use the traditional assembly method, this will bring a huge amount of work. Due to the low efficiency of traditional assembly, it will seriously delay our experimental process. And Gibson assembly can't guarantee the success rate, and it is not suitable for our large amount of work. Therefore, after the discussion, we finally decide to use GoldenGate Assembly. However, the selection of the correct construct is also a problem, so we designed the following Construction Device to select the correct constructs by visible color changes of the colonies.

    The Construction Device includes a promoter J23104, a pigment protein tspurple, a pigment protein cjblue, a BbsI site, and a BsaI site. J23104+cjblue can be replaced with other promoter sequences by using BbsI restriction enzymes, and tspurple can be replaced with RNA thermosensor sequences using BsaI restriction enzymes. When not replaced, the colonies appear blue due to the expression of cjblue. After the first replacement, the colonies of the correct constructs appeared purple. After the second replacement, the colonies of the correct constructs exhibited their own milky white color. Color changes can refer to the following table:

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

  • References

    • Dai H, Wang Y, Lu X, et al. Chimeric antigen receptors modified T-cells for cancer therapy[J]. JNCI: Journal of the National Cancer Institute, 2016, 108(7).
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