Team:MIT/Contribution

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Parts Characterization and Improvement

Parts Characterization:

Basic Parts: BBa_K1119006/BBa_I712004 (CMV) and BBa_K2520023 (hEF1a)

While the majority of our project was focused on engineering leader cells, we were also interested in manipulating the follower cells by genetically engineering HL-60 cells. We noticed that while there were several methods of transfection described for HL-60 cells ( including a project by the 2009 UCSF iGEM team, a paper by Park et. al.), we did not find any systematic data on the function of commonly used promoters in this cell type. Considering that HL-60 cells are relatively difficult to transfect and require harsh transfection conditions (electroporation) that can result in cell death and low transfection efficiency, we wanted to find a promoter that would lead to reliable and strong expression of transfected genes in order to facilitate our future experiments with the SynNotch system and engineering of leader cells to become followers.

In particular, we characterized the expression of the fluorescent proteins EYFP and TagBFP encoded on plasmids under the CMV and hEF1a promoters and transfected by electroporation into undifferentiated HL-60 cells.

We could not identify the exact CMV promoter that matched up to ours, as there are multiple copies of similar sequence/related function: http://parts.igem.org/Part:BBa_K1119006 or http://parts.igem.org/Part:BBa_I712004. We also could not find the exact hEF1a promoter that matched up to ours but decided this was similarly used/close enough to our hEF1a promoter: http://parts.igem.org/wiki/index.php/Part:BBa_K2520023

We observed a lot of cell death due to electroporation. Events from the flow cytometry analysis were first plotted on an FSC/SSC dot-plot graph to set an analysis gate, as shown in Figure 1. For the cells within the analyzed gate we looked at fluorescence in the FITC channel (excitation 488 nm, detection window 530/30 nm) for detection of EYFP and Pacific Blue channel (excitation 405 nm, detection window 450/50 nm) for detection of TagBFP. We found that 52% of cells transfected with CMV-EYFP were fluorescent in the yellow channel, and 35% of cells transfected with CMV-TagBFP were fluorescent in the blue channel. On the other hand, only 1.5% of cells transfected with hEF1a-EYFP and 8% of cells transfected with hEF1A-TagBFP were weakly fluorescent.

Figure 2 shows the overlay of histograms for untransfected cells (control, shown in green) and cells transfected with the fluorescent protein encoded under a CMV promoter (shown in blue) or hEF1a promoter (shown in red) for a) TagBFP and b) EYFP.

Figure 1:


Link to parts page

Figure 2a:


Link to parts page

Figure 2b:


Link to parts page

Parts Improvement:

Basic Part: BBa_K1761003

We improved mNeonGreen (BBa_K1761003) by using it as a fusion protein tag and attaching it to the IL-8 gene in the form of BBa_K2957002 (IL8 with NeonGreen tag). We believe we improved this part for the following reasons:

  1. We improved this part by proving its use as a fusion fluorescent protein tag to the IL-8 chemokine and CCL5 chemokine. This allows it to visually be tracked in the cell.
  2. We proved that it can be secreted out of the cell carried by a chemokine with a natural secretion tag. (see secretion results on composite parts page, BBa_K2957007)

This page was written by Jan Lonzaric, Melody Wu