Team:RHIT/Demonstrate
In this project, we successfully produced two yeast expression vectors with BioBrick prefix and suffix. Using the vectors we produced, we managed to verify function of mitochondrial localization signal (mls) by testing the hybrid construct mls-yeGFP under a fluorescence microscope. Unfortunately, experimental results indicated that expressing the mitoribosomal protein mRPS12 in a haploid mRPS12 knockout strain did not restore aerobic respiration and their ability to grow on non-fermentable carbon sources. We replicated this experiment several times while the same results were obtained. Expressing mRPS12 in wild type S. cerevisiae did not result any adverse effect, therefore we can conclude that its failure to restore mitochondria function in the knock out strain did not result from overexpression of the gene behind the GPD promoter. Therefore we hypothesize that the knock out strain we used has lost its mitochondrial genome which explains why restoring mitoribosomal function does not restore the strains ability to process glycerol. We continued our investigation using Janus Green B and the results indicated that…
Although the mRPS12 verification experiment failed this time, we are still positive about the hypothesis we have about it. In the future, we can replicate this experiment using a more recent mRPS12 yeast knockout strain to avoid the possibility of losing mitochondria. We will transform the yeast strain with our pSB416-GPD vector with an additional copy of mRPS12 TU as insert and replace the genomic mRPS12 TU gene with a Kan Mx cassette. We can then test mRPS12 TU function by using plasmid shuffling with an empty pSB413-GPD Vector and the pSB416-GPD vector with mRPS12 TU to show that removing and restoring mRPS12 TU can turn aerobic respiration on and off in yeast.
Once the function of mRPS12 is verified, we would like to connect a light-switchable system [1] to the mRPS12 gene in order to achieve remote control. Essentially the system utilizes protein-protein interaction between phytochrome B (PhyB) and phytochrome interacting factor 3 (PIF3) to control gene expression. By attaching a chromophore phycocyanobilin (PCB) extracted from cyanobacterium to PhyB, exposure to red light would change conformation of PhyB from ground state Pr to active state Pfr. Such conformation change would alter interaction between PhyB and PIF3, resulting in turning the following gene on or off. This system is originally proposed in 2002 and it was based on Gal4 promoter [1], while an iGEM team from Munich modified it by replacing Gal4 promoter with LexA [2]. The advantage of using LexA is that, since LexA comes from prokaryotes, it minimizes the interference between endogenous yeast metabolism [2].
