Part:BBa_K2077002
mls-yeGFP
According to 2015 Rose-Hulman iGEM team, the coding sequence of mitochondrial ribosomal protein mRPS12 starts with a mitochondrial localization signal (mls). From previous research, it is possible that this mls protein is able to transport and direct any attached protein into the mitochondria. To verify the hypothesis brought up in 2015, we constructed a hybrid system with the mls protein and a reporter protein. Instead of using the regular GFP, yeast-enhanced green fluorescent protein (yeGFP) was used in this system as optimal gene expression can be achieved.
Figure 1: Observation of S. cerevisiae yeast cells that contain mls-yeGFP construct (test) with a 60X oil immersion objective. The right picture shows yeast cells under bright-field microscopy. The middle picture shows yeast cells under fluorescence microscopy. The left picture shows the overlay of the above mentioned two pictures.
Figure 2: Observation of S. cerevisiae yeast cells containing empty pSB416 GPD plasmid (negative control) with a 60X oil immersion objective. The right picture shows yeast cells under bright-field microscopy. The middle picture shows yeast cells under fluorescence microscopy. The left picture shows the overlay of the above mentioned two pictures.
Figure 3: Observation of S. cerevisiae yeast cells that contain mls-yeGFP construct (test) with a 60X oil immersion objective. The right picture shows yeast cells under bright-field microscopy. The middle picture shows yeast cells under fluorescence microscopy. The left picture shows the overlay of the above mentioned two pictures.
Figure 4: Comparison between Mito ID Red florescence, left, and mls-yeGFP florescence, right, in the mRPS12 TU knock out yeast strain.
Figure 5: Comparison between Mito ID Red florescence, left, and mls-yeGFP florescence, right, in the mRPS12 TU wild type yeast strain.
As shown in Figure 1 &3, scattered green fluorescence was observed inside some yeast cells. According to the pattern of fluorescence, we are able to conclude that GFP is targeted to some internal organelles rather than cytoplasm, and it is possible that the fluorescence comes from mitochondria. However, additional experiments with mitochondria fluorescence dye is needed to show the actual location of mitochondria in order to fully prove the function of mls. Comparing to Figure 1, the negative control (Figure 2) does not emit any fluorescence under the same excitation condition, indicating that empty pSB416 GPD plasmid did not affect mls-yeGFP expression. A blown-up image of fluorescing yeast cells is shown below in Figure 3. It clearly shows that the intensity of fluorescence varies throughout the cells, indicating mls protein is targeting GFP to certain regions of the cell, which possibly would be the mitochondria. The targeted fluorescence pattern of mls-yeGFP matches the fluorescence pattern of mitochondria targeting red fluorescent stain Mito ID Red, verifying that the mRPS12 mls preprotein peptide does target proteins to mitochondria (Figures 4 & 5). Additionally mRPS12 mls targets proteins to the mitochondria regardless of whether or not a yeast strain can translate its mitochondrial genome and/or use its electron transport chain for respiration, as illustrated by the lack of difference between fluorescence pattern similarities between the red stain and mls-yeGFP in both an mRPS12 TU, BBa_K1729001, knock out strain that has mitochondria that lack a functional electron transport chain and mitochondrial ribosomes, and the wild type strain with functional mitochondria (Figures 4 & 5).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 764
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