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:

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.

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.

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.

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.

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.