The double hybridization process requires two conversions, which is a considerable amount of work, especially when looking for new proteins to act on.Moreover, yeast cells are about four orders of magnitude less efficient at transforming than bacteria.Therefore, the transformation step becomes the bottleneck of dual hybridization technology.Bendixen et al. avoided two conversion operations and improved the efficiency of double hybridization by citing yeast conjugation type.In the sexual reproduction process of yeast involves two kinds of mating types: a mating type and a mating type, this two kinds of haploid mating (mating) can form a diploid, but a mating type of cells or a mating type of cells can not form a diploid.According to the characteristics of yeast sexual reproduction, they transformed library plasmids into alpha-zygotic yeast cells, and "decoy" expression vectors into a-zygotic cells.Then the screen plate was respectively applied to make the cells grow into lawn, and then the two kinds of mycorrhizae were copied onto the same triple screen plate. In principle, only diploid cells with interaction between decoys and target proteins could grow on this plate.Haploid cells or diploid cells that do not interact with DB fusion protein and AD fusion protein are eliminated.The resulting clones were further identified by activity of beta-galactosidase.This improvement not only simplifies the experimental operation, but also improves the screening efficiency of dual hybridization.
Vidal et al. developed the so-called reverse two-hybrid system.The key to this technique is to report the introduction of the gene URA3.The URA3 gene plays an anti-selection role here, encoding enzymes that are key to uracil synthesis.This enzyme converts 5-fluorowhey acid (5-foa) into a cytotoxic substance.Vidal et al. introduced the binding site of Gal4 into the promoter of URA3 gene by modifying it.This modified yeast strain can only grow in a selective medium lacking uracil if the "bait" and "prey" interact to activate the URA3 gene expression.Interactions between "bait" and "prey" inhibited cell growth in a complete medium containing 5-foa.However, if the target protein, that is, the protein fused with DB or AD, mutates or the URA3 gene is no longer expressed due to additional drug interference, the cell can grow in a complete medium containing 5-foa.In this way, Vidal et al. screened mutants of transcription factor E2F1, which still bind to retinoblastoma protein RB, but lost the ability to bind to another protein called DP1.Results the results were verified by in vitro binding experiments.By sequencing the genes of these mutant proteins, they found new sites where E2F1 binds to DP1.
In some cases, beads that do not grow well in liquid medium may grow well in solid medium.If the decoy protein is toxic to yeast cells, transcriptional activation domains can be cut off through gene recombination and then retested for self-activation, but note that recombination may also disrupt interactions between proteins.One or even two fusion proteins are toxic to yeast cells.You can reduce toxicity by recombinant methods while still ensuring protein interactions.Or use a vector with a lower expression level.Hybridization can also be performed on AGAR plates or filtration membranes.But at the same time a cross - control experiment must be carried out.The conversion efficiency of yeast two-hybrid experiment is too low, which can be solved by the following methods:
1) check the purity of DNA and, if possible, repurify it with ethanol.
2) dna-bd/decoy protein is likely to be toxic.
3) for inappropriate medium, remix the medium and conduct control transformation.
4) test the conversion efficiency of pGBT9 control vector. Place it in the SD/ -- Trp plate, and the conversion efficiency should be more than 1 x 105 colonies/mg DNA.
In hybridization, the number of pre-transformed decoy cells may be insufficient and the hybridization efficiency is not high.Large, fresh clones should be selected for culture after overnight liquid culture of the decoy strain. After centrifugation and resuspension, the cells should be counted using a hematometer.
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