Team:Hong Kong HKUST/characterization/mls

Introduction

Mitochondrial Leader Sequence (MLS) helps direct proteins to the mitochondria when this peptide sequence is in front of the N-terminus of the protein of interest. MLS will be removed upon the peptide’s translocation into the mitochondria, but four additional amino acid residues (Ile-His-Ser-Leu) will be left at the N-terminus of the protein. The CDS of MLS was cloned out from pCMV/myc/mito (Invitrogen, Carlsbard, CA) using PCR.

MLS is submitted in RFC25 standard (BBa_K1119001) to facilitate fusing with other CDS. MLS in RFC10 standard (BBa_K1119001)is submitted as alternative. but cannot be fused directly to other CDS due to limitations in RFC10. Users who obtained the part in RFC10 standard can obtain the part by PCR and fuse to other domains using Splicing by Overlapping PCR.

In our characterization, the CDS of MLS was assembled in frame with that of GFP reporter using Freiburg’s RFC25 format(BBa_K648013). The translation unit was driven by CMV promoter (BBa_K1119006) and terminated by hGH polyA signal (BBa_K404108). The MLS-GFP generator (BBa_K1119009) was then transfected into HEK293FT cells. Mitochondria were stained after transfection and co-localization was determined by area of signal that overlapped. To provide a positive control, CDS of EGFP from pEGFP-N1 (Clontech) was inserted downstream and in frame with the CDS of the MLS in the commercial plasmid pCMV/myc/mito, (Invitrogen, Carlsbard, CA). A negative control was made by GFP generator that does not contains the CDS of MLS (BBa_K1119008).

The detailed protocol of our characterization can be found in HKUST iGEM 2013 Wiki.

Cell Culture and Transfection

The three constructs were transfected into different batches of HEK 293FT cells separately. The protocol for culturing HEK 293FT cells was based on ATCC. The culture medium used was DMEM with 10% FBS and 1% penicillin/streptomycin. For the transfection, we have followed the general procedure outlined by Invitrogen LipofectamineTM 2000. Serum free and antibiotics free DMEM was used to form DNA-lipofectamine complex.

Stain with Mitotracking Dye

In order to test the efficiency of MLS, we need to identify the location of mitochondria. Therefore, rosamine-based MitoTracker® Red CMXRos (Invitrogen, Carlsbard, CA), which is a red-fluorescent dye, was used to stain the mitochondria in live cells. It can accumulate due to the membrane potential of mitochondria. According to the manual, this dye can be well resolved from the green fluorescence of other probes so it won’t affect the observation of the expression of green fluorescence protein. The protocol we used for staining is from MitoTracker® Mitochondrion-Selective Probes. The working concentration of staining solution made was 0.2μM and the incubation time after staining was 15 minutes.

Fluorescence Microscopy

Since the EGFP, GFP and MitoTracker® Red CMXRos can all be observed under fluorescent microscope, we did fluorescent microscopy in order to obtain results. We observed the transfected cells under confocal microscope.

Data Processing

To quantify the amount of signal overlapped between the GFP signal and the red fluorescence signal from MitoTracker® Red CMXRos, we adopted the method described by A.P. French et al. in “Colocalization of fluorescent markers in confocal microscope images of plant cells” (French et al., 2008). With the use of Pearson-Spearman correlation colocalization plugin for ImageJ, scatterplots of the green intensities (y-axis) and red intensities (x axis), Pearson's correlation coefficient and Spearman correlation coefficient were generated.

Result

Figure 1. MLS directs GFP into mitochondria. When MLS is added to the N terminus of GFP, the GFP was directed to the mitochondria in the cells, giving patches of GFP signal that overlapped with the signals from MitoTracker®. When MLS is not added to the GFP, the GFP signal can be seen scattered all around in the cell. Scale bar = 10 microns

Figure 2. Scatter plots of fluorescence intensities of green (y axis) and red (x axis) from images shown in Figure 1. It showed that the BioBrick MLS-GFP and commercial GFP construct had linear relationship of green intensities and red intensities while the GFP generator had no relationship. Pearson's correlation coefficient (rp) and Spearman correlation coefficient (rs) were determined using the Pearson-Spearman correlation colocalization plugin (French et al., 2008) for ImageJ with a threshold of 0 and listed for each image.

Figure 3. Mean Pearson correlation coefficient(rp) and mean Spearman correlation coefficient(rs) were shown in bar chart. Using ImageJ software and plugins, the Pearson correlation coefficient and Spearman correlation coefficient were generated. For every batch of transfected cells, four samples were used for quantification. Experimental BioBrick MLS-GFP and commercial MLS-GFP: Coefficients were close to 1, good colocalization ; GFP: Coefficients were close to 0, poor colocalization. Error bars show standard deviation.

Conclusion

The result shows that the colocalization of GFP signal and mitotracking dye is high for the characterization construct of MLS BioBrick. It can be concluded that the MLS can function well and successfully bring recombinant protein into mitochondria.

Reference

pcmv/myc/mito Invitrogen.(2012).pShooter™ Vector(pCMV/myc vectors).Retrieved from http://tools.lifetechnologies.com/content/sfs/manuals/pshooter_pcmv_man.pdf
Dean, J., Tran , L., Liao, J., Dipple, K., Reue, K., Tontonoz, P., & Beaven, S. (2009). Resistance to diet-induced obesity in mice with synthetic glyoxylate shunt. Cell Metabolism, 9((6)), 525–536. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/19490907
French, A.P., S. Mills, R. Swarup, M.J. Bennett, and T.P. Pridmore. (2008). Colocalization of fluorescent markers in confocal microscope images of plant cells. Nature Protocol. 3:619–628. doi:10.1038/nprot.2008.31.