1. Using two different Y2H approaches [def See Support Pg. 1 for more info.], the study determined a total of 2,045 binary interactions between 820 proteins of S. pneumoniae. All available proteins were screened against each other sing a Gal4-based Y2H system.
2. 360 hypothetical proteins were screened using a LexA-based system.
3. A set of 322 protein interactions were determined using a microfluidic high-throughput assay.
This microfluidic assay consists of a microfluidic chip that consists of multiple micro-channels that interacted and flowed with the cDNA of two interacting proteins. Two proteins only were chosen in order to test the binary interactions between them. [3 Meier M, Sit RV, Quake SR. 2013. Proteome-wide protein interaction measurements of bacterial proteins of unknown function. Proc Natl Acad Sci U S A 110:477–482. .].Bait and prey proteins of interest were fused to a short sequence of either the DNA binding domain (bait) or the activation domain (prey). This then results in the process of transcriptional fusion which allows this fusion to place a reporter gene downstream of the protein's promoter. Transcriptional activity is then measured with the promoter [def A region of DNA that allows for transcriptional processes for a particular gene].
Combining the results of these three data sets resulted in having 43% of the S. pneumoniae proteome covered. The image on the left shows the cultivation of the interactome of the proteins that were found for S. pneumoniae.
Proteins with known functions are colored green and the unknown functions were colored red. Roughly 37.2% of the proteins in the network were characterized to be unknown. Y2H translates to yeast two-hybrid
and this identifies the proteins in the interactome that have undergone this process. Microfluidic is the other process (as shown as dark grey) and identifies the proteins in the data set for method 3 as listed above.
