Gibson Assembly and Yeast Assembly
Following the production of our GFP gene by templateless and finish PCR and verification that most of this DNA is the correct size by gel electrophoresis, we combine the gene with a promoter (RNR3) and a plasmid vector. Traditionally, we would clone the PCR product into the vector by using restriction enzymes to cut each piece of DNA and then we would join them together with the enzyme DNA ligase. Instead of the traditional method, we will use a newer method called Gibson assembly. Gibson Assembly was developed by Dr. Daniel Gibson and his colleagues at the J. Craig Venter Institute. It allows for successful assembly of multiple DNA fragments, regardless of fragment length or end compatibility (the presence of compatible sites for restriction enzyme cutting). It has been rapidly adopted by the synthetic biology community due to its ease-of-use, flexibility and suitability for large DNA constructs.
The Gibson Assembly Master Mix contains enzymes and therefore should be kept ON ICE AT ALL TIMES!
1. Label a 1.7 ml microcentrifuge tube with your initials.
2. In that tube (on ice), combine:
Promoter 3 ul Vector 3 ul GFP gene 3 ul Gibson Assembly Master Mix 10 ul
2. Place tube in a water bath and incubate at 50C for 15 minutes.
3. Remove tube from water bath and store at -20C until bacterial transformation in 2 weeks.
Although we have just used enzymes to assemble our GFP gene with a promoter and a plasmid vector (Gibson Assembly), the process of recombination, and therefore chunk assembly, is much more efficient in yeast cells. Therefore, we will also assemble our DNAs in yeast to compare the efficiency of transformation. Note that we will need to use a different plasmid vector for yeast assembly. Plasmid vectors typically contain the ampicillin resistance gene so that bacterial cells containing the vector will be able to grow in ampicillin. The plasmid we will use today will also contain a selectable marker so that yeast cells containing the vector will be able to grow on media lacking the amino acid histidine.
1. Prepare a yeast transformation master mix and keep the mix on ice. You have a tube containing 960 ul of 50% polyethylene glycol (PEG). To this tube add 144 ul of 1.0M lithium acetate (LiAc), 40 ul of single-stranded herring sperm DNA, and 55 ul water. Mix very well by vortexing for 10 seconds.
2. Obtain three tubes of yeast competent cells (one for assembly of the GFP gene/promoter/vector, one for the positive control, and one for the negative control). Label these tubes “GFP”, “PC”, and “NC”.
3. Centrifuge the yeast cells at 13,000g for 1 minute at room temperature.
4. Remove the supernatant (the liquid) with a pipet set to 1000 ul. Discard the liquid.
5. Aliquot 286 uL of yeast transformation mix from step 1 into each of the three microcentrifuge tubes containing the yeast cell pellets. Pipet up and down to mix the yeast cells.
6. To your “GFP” tube, add 3 ul of the GFP gene, 3 ul of the promoter, and 3 ul of the vector.
7. To the “PC” tube, add 9 uL from the provided PC DNA tube (this is 1 ug of the circular plasmid).
8. To the “NC” tube, add 9 uL of sterile water.
9. Vortex the tubes for 10 seconds to thoroughly mix the DNA with the transformation mix and yeast cells.
10. Incubate in a 42°C water bath for 20 minutes. During this time, obtain 3 SC-His plates. Label one “PC”, one “NC”, and one “GFP”.
11. After the heat shock, centrifuge the three tubes at top speed for 30 seconds.
12. Remove the supernatant with a P1000 pipette set to 1000 ul and discard the liquid.
13. Add 250 μL of sterile water to each tube. Gently pipette to resuspend the pellet.
14. From each tube, transfer 250 ul of the transformation product onto your appropriately labeled SC-His plates and spread to distribute evenly.
15. Once the plates are no longer wet, incubate the plates at 30°C for 2 days (remember to turn the plates upside down).