Team:Jilin China/Construction
Construction
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Golden Gate Assembly
RThis year, we had hundreds of measurement devices to construct, which means that we need a efficient method to reduce the time and cost of construction. To address this issue, we chose the Golden Gate assembly , which has the advantages of short time-consuming, simple operation and high success rate.
This assembly method is based on the Type IIS restriction endonucleases such as BsaI and BbsI, witch differ from Type II restriction enzymes in several ways( figure 1). There are several advantages to use Type IIS restriction endonucleases.
1. The recognition sequences of Type IIS restriction endonucleases comprise two distinct sites, one for DNA binding, the other for DNA cleavage. DNA would be cleaved at a defined distance from their non-palindromic asymmetric recognition sites, creating 4-base overhangs. Therefore, If the Type IIS recognition site is 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 Golden Gate assembly.
2. 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 DNA molecules generally have different overhangs, therefore, and will not anneal to one another. However, if the sequences of the overhang are predetermined, by designing them into oligos, then it can be made to complement another and to be directional. Then the DNA fragments can be stitched together in the correct order and orientation in a single ligation.
R II
R IIS
Figure 1. The difference between the Type II restriction edonuclease and Type IIs restriction edonuclease.
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Design of the construction immediate and inserts
Firstly, in order to get the measurement device with suitable promoters and different RNA-based thermosensors, we designed the construction immediate (figure 2) in plasmid backbone pSB1C3, which are used to be ligated with many different fragments gene of interest. For the ‘scar-less’ assembly, we add the recognition sites of type IIS enzymes on the construction immediate, which will be digested by enzyme and will not appear at the end constructs. The fragments gene of interest contain complementary sticky ends can ultimately be assembled by simple ligation.
Figure 2. The construction immediate.
Secondly, our fragments gene of interest such as the DNA fragments of promoters and RNA-based thermosensors contain complementary sticky ends, witch can ultimately be assembled into the construction immediate by simple ligation.
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The workflow of the construction and selection
There are stage A, stage B and stage C to construct the measurement devices and selected them by the difference in color.
In stage A, the construction immediate will express chromoprotein cjBlue in E.coli. We used Type IIS restriction enzyme BbsI to cut the Part I that contains the sequences of original promoter, cjBlue and double terminator (figure 3, (1)). Then we ligated the inserts of promoters with different expression strength on the plasmid and got the Stage B (figure 3, (2)).
If the previous steps were successful, the plasmid could express chromoprotein tsPurple rather than cjBlue in stage B. At this time, We chose the purple monocolony and extracted its plasmids,.After the correct sequencing, then we conducted the next step.
In the next step, we used Type IIS restriction enzyme BsaI to cut the Part II that contains the sequences of RBS, tsPurple and double terminator (figure 3, (3)), and replaced this region with the inserts of different RNA-based thermosensors (figure 3, (4)).
If the previous steps was successful, the plasmid could express superfold GFP rather than tsPurple. After the correct sequencing, we got different measurement device. If they did not have expression strength that is easy to detect, we would replace the promoter until the appropriate intensity was reached.
Figure 3. The steps of construction of measurement devices.
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The procedure of Golden Gate assembly
We followed three steps to assemble many measurement devices as follows:
1.Annealed oligos
This step could be use to add any short DNA fragment to a plasmid. Most of the thermosensors we designed are 40~80bp, so we designed top oligos with the sequences of thermosensors and with the bottom oligos, which would be the reverse compliments so that they could anneal. We made sure our annealing products contain the cohesive end bases to complement the overhangs on the construction device (Figure 4).
Figure 4. Diagram of Annealed oligos
2. Phosphorylation
After annealing, the 5' phosphorylation of DNA was required for the subsequent ligation. We phosphorylated fragments with T4 Polynucleotide kinase, which could catalyze the transfer and exchange of P from the γ position of ATP to the 5'-hydroxyl terminus of polynucleotides and 3'-monophosphates (Figure 5).
Figure 5. Diagram of Phosphorylation
3.Digestion and Ligation
We chose Type IIS restriction enzyme to digest the plasmid and used T4 DNA ligase to assemble DNA fragments we had phosphorylated before (figure 6).
Figure 6. Diagram of Digestion and Ligation
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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.