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Characterizations

Mitochondrial Leader Sequence (BBa_K1119000, BBa_K1119001)

Introduction

In our characterization, the coding DNA sequence (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 aforementioned construct (BBa_K1119009) was then transfected into HEK293FT cells. Mitochondria were stained with MitoTracker® Red CMXRos dye after transfection and co-localization between the GFP signal and that of the dye was determined as the area of signal overlap. To provide a positive control, the 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). Our negative control construct was the same as our experimental construct, but minus the MLS CDS. (BBa_K1119008).

MLS is submitted in RFC25 standard (BBa_K1119001) to facilitate fusing with other CDS. MLS in RFC10 standard (BBa_K1119000) is submitted as alternative but it cannot be fused directly to other CDS due to limitations in RFC10. Users who obtained the part in RFC10 standard can amplify the part by PCR and fuse it to other domains using overlapping PCR.

To quantify the amount of signal overlapped between the GFP signal and the MitoTracker® dye, 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 the Pearson-Spearman correlation colocalization plugin for ImageJ image processing, scatter plots of the green intensities (y-axis) and red intensities (x-axis), Pearson's correlation coefficient and Spearman's 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 inside the cell. Scale bar = 10µm.

Figure 2. Scatter plots of fluorescence intensities of green (y-axis) and red (x-axis) by construct. It showed that the BioBrick MLS-GFP and commercial MLS-GFP construct had a linear relationship of green intensities and red intensities while the GFP generator alone had no relationship. Pearson's correlation coefficient (rp) and Spearman's 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. Calculated mean Pearson's (rp) and Spearman's (rs) correlation coefficients for each construct. The coefficients were generated using ImageJ software and specific plugins. 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.

CMV Promoter (BBa_K1119006)

Introduction

For this promoter's characterization we assembled it with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108). The Pcmv-GFP was then transfected into HEK293FT cells and the in vivo green fluorescence signal was observed under confocal microscope. The positive control was pEGFP-N1 (Clontech) that contains CMV promoter and EGFP reporter. The negative control was the same as the experimental construct, but minus the promoter. The detailed protocols employed for our characterization work can be accessed through the link.

EF1-alpha Promoter (BBa_K1119004)

Introduction

The constitutive Human Elongation Factor-1alpha (EF-1alpha) promoter regulates gene expression in mammalian cells. Up to now, only P_cmv has been used widely as a constitutive mammalian promoter in the iGEM competition. Here we introduce the EF-1alpha promoter that is known to be a consistently strong promoter in many cell types. The origin of this part is the Homo sapiens chromosome 6 genomic contig, GRCh37. p13.

For the characterization of this part, the DNA sequence of EF-1alpha promoter was assembled with GFP reporter (BBa_K648013) and hGH polyA terminator (BBa_K404108) using Freiburg’s RFC25 format. The EF-1alpha promoter-GFP was then transfected into HEK293FT cells and in vivo green fluorescence signal was observed under fluorescence microscope. The positive control was iDUET101a plasmid (Addgene plasmid 17629) that contains EF-1alpha promoter and EGFP reporter. Our negative control was the same as the experimental construct, but without the EF-1alpha promoter. EF-1alpha promoter efficiency was compared with that of the CMV promoter by also transfecting a GFP reporter driven by CMV promoter (BBa_K1119006) and terminated by hGH polyA signal (BBa_K404108). Detailed protocols for our characterization work can be accessed via the link.

Result

Figure 1: No GFP signal of EF-1alpha promoter was observed. While cells transfected with iDUET101a (positive control) and pCMV-GFP (comparison construct) showed GFP signal, those transfected with EF-1alpha promoter-GFP did not give GFP signals. Our negative control, GFP without promoter did not gave any GFP signals. Scale bar = 10µm.

Conclusion

The sequence of EF-1alpha promoter cloned from iDUET101a contains the full sequence of the functional promoter region labeled in pBudCE4.1 (Invitrogen). We believe that EF-1alpha triggers transcription of GFP but fails to translate the GFP coding sequence due to having a too short 5’ untranslated region. Additional junk sequences should be added before the first start codon to elongate 5’ untranslated region, which may lead to more successful translation.