YaliFunTome
Yarrowia lipolytica Functional Screens of Tanscription Factor-ome Database
Experimental setup
In short: A collection of 125 Y. lipolytica strains over-expressing (OE) individually TFs was subjected to extensive phenotype screening under different environmental conditions, and the data processing was assisted by mathematical modeling. It was presumed that apart from genetic engineering of the TFs-encoding genes by their OE, implementation of the environmental perturbations will further manipulate the activation status of the TFs.
The search of the TFs within the Y. lipolytica genome and construction of the strains co-over-expressing (co-OE) individually one of 125 identified TFs and a reporter protein (RedStar2) was done previously, by our Colleagues: doi:10.1093/femsyr/foy037. Both genes were cloned under a constitutive promoter pTEF (more details can be found here: doi:10.1093/femsyr/fov052).
The TF co-OE strains were cultured under a combination of enviromental conditions. The experimental setup was designed using DoE (Design of Experiments; DesignExpert software; StatSoft), as presented below:
Tab.1. Summary of experimental setup designed with DoE (DX. StatSoft).
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Design Type: Face-centered Central Composite Design |
Runs: 104 for each of 125 TF = 13 000 in duplicate = 26 000 readouts on FL (fluorescence) and growth (OD600) |
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| The central variants were repeated five times. The order of culture execution was randomized. | ||||||||||
| Process Factors | Categoric: A, B, C, Numeric: d e | |||||||||
| Factor | Name | Units | Change | Type | Minimum | Maximum | Coded Low | Coded High | Mean | Std. Dev. |
| A | AIR "limiting" | 0/1 | Easy | Categoric | low | high | Levels: 2 | |||
| B | Carbon source | Glu/Gly | Easy | Categoric | Glucose | Glycerol | Levels: 2 | |||
| C | Nitrogen source | i/o | Easy | Categoric | Inorganic: Ammonium Sulfate |
Organic: Casamino Acid Hydeolysate |
Levels: 2 | |||
| d | pH | pH | Hard | Numeric | 3 | 7 | -1 3 | +1 7 | 5 | 2.83 |
| e | Temperature | °C | Hard | Numeric | 22 | 34 | -1 22.00 | +1 34.00 | 27.1 | 5.443 |
Since typical multitier plates were found useless for reliable high-throughput screens and phenotype reads of Y. lipolytica strains, we used 24-well Duetz-System plates, covers, and clamps; in 2.5 mL working volume.
Fig.1. Graphical presentation of the arrangement of the variables' combinations corresponding to the data presentation scheme (left), and the variables' coding system (right). CH - casamino acid hydrolysate, AS - ammonium sulfate, OA- oxygen availability, FC - fold change of the response from the TF-OE strain over the control strain .