Team:BIT-China/Project/Transduction
Transduction
In order to enhance the signal transmission and amplification, we need to knock out some genes of yeast, including Ste2, Sst2 and Far1. Ste2, Sst2 and Far1 exist in endogenous GPCR pathways of yeast, which responds to the GPCR signal and start the downstream of the Signaling pathways. Ste2 interferes with the sweetness signals which respond to the pheromone. Knocking out Sst2 is necessary to remove the inhibition of sweetness signals. Far1 regulars the cell division which may have a bad influence on the yeast after the genes are knocked out. All in all, knocking out these genes is an essential step of the experiment. The yeast can cause the gene replacement or gene lost by homologous recombination, for that reason these genes will be knocked out by the means of designing the primers cleverly.
To make our yeast detect sweeteners, we replace ste2 receptor with Human sweet receptor T1R2-T1R3. So, first, we have to knock out ste2 gene. To decrease other interference factor we knock out sst2 gene and far1 gene. sst2 and far1 are genes that functioned to tunning signals among the MAPK pathway, they are believed to coordinated growth while yeast’s mitosis as described in the Design.
By NCBI databases for obtaining ste2 sst2 and far1 gene sequence information, we find appropriate homologous arm in upstream and downstream genes. In order to knock out these genes effectively, we select different lengths of homologous arms, including 50bp, 200bp and 500bp. According to the wet-lab measurement, we find out 200bp and 500bp homologous arms are more efficient.
We design 3 pairs of primers cleverly whose templates are the upstream homologous arm, marker and the downstream homologous arm respectively. The markers are Histone synthesis gene, Uracil synthesis gene and Trptophan synthesis gene, short termed His, Ura and Trp . His will replace sst2, Ura will respanlace far1 and Trp will replace ste2. Initially, we get 3 fragments by PCR and they have the overlap areas with each other.
Secondly, the complete fragment observed by OE-PCR is converted to the yeast which has been already knocked out the endogenous gene Uracil, Histone and Trptophan. Additionally, the colony is chosen on the relevant nutritional deficiency medium, so that only the positive cloning can survival on it.
To verify whether the gene is actually knocked out and avoid the false positive colonies, we design the primer1,2,3 and 4 for each genes, as shown in the figure1. The primer 1 and primer 4 are on the yeast genome. The primer 2 is on the marker and primer 3 is on the gene which will be knocked out.
If we get correct results by the primer 1 and 2 as well as nothing from primer 1 and 3, demonstrate that the gene is knocked out. Then we sequencing the PCR product using primer 1 and 4 to make sure the sequence is right.click here to read results
After knocking out far1, we test the growth curve of the yeast. Far1 protein is a cyclin-dependent kinase inhibitor. Deletion far1 gene can relieve inhibition. As you can see, it’s……
After knocking out sst2, we test the growth curve of the yeast. Sst2 protein is an important negative regulatory factor of GPSTP. When we knock out sst2 gene, the sensitivity of the yeast GPSTP could be improved. And the inhibition of cell growth will be enhanced.
As you can see, ΔSst2 strain is much more sensitive to α pheromone. Compared with CENPK2-1C strain, less amount of α pheromone can cause yeast growth arrest.
