Difference between revisions of "Part:BBa K2520023"

Latest revision as of 03:40, 22 October 2019

EF1a promoter

Human elongation factor-1 alpha (EF-1 alpha) is a constitutive promoter of human origin. It can be used in-vitro and in-vivo to induce ectopic expression of recombinant genes. This promoter is very useful in cells where other promoters, such as CMV, are underactive or silenced (such as stem cells).

Previous usage in iGEM

The EF-1a promoter was used by both teams that won “Best Therapeutics Project” last year (http://2016.igem.org/Team:Tel-Hai, http://2016.igem.org/Team:SYSU-MEDICINE"). This promoter allows for strong and constitutive expression in a wide variety of cell lines, and is especially useful when working with stem cells. This promoter has hitherto not been added to the iGEM parts registry as it included two forbidden restriction sites in its sequence. Since promoters are not translated, the base pair sequence cannot simply be changed through silent mutations.

Since this promoter is clearly very important in iGEM, and specifically for therapeutics, we decided to mutate the original (WT) EF-1a at two points (base pair 319 and 824 were changed from C to T), thus eliminating the forbidden restriction sites. We characterized and tested our new promoter for functionality and compared it with the WT EF-1a (Figure 1). In closing, we have added a new and invaluable promoter to the iGEM part registry that we believe will serve future teams seeking to work with mammalian cells.

Results

In order to test our mutant promoter, we tested the expression of the reporter gene GFP under three different promoters- CMV, EF1a WT and the mutant EF1a we created. As shown in the following graph, the expression levels of the reporter gene under EF1a promoter was much higher as compared to the CMV promoter, and these levels are almost equal between the WT and the mutant promoter.

Figure 1: Fold change of Fluorescence intensity of GFP under three different promoters- CMV, WT EF1a and mutant EF1a, compared with non-transfected cells, using Flow Cytometry, FITC channel.

References

Mikkola, Hanna, et al. "Lentivirus gene transfer in murine hematopoietic progenitor cells is compromised by a delay in proviral integration and results in transduction mosaicism and heterogeneous gene expression in progeny cells." Journal of virology 74.24 (2000): 11911-11918.‏ APA

MIT-2019 Characterization

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 <a href=”http://2009.igem.org/Team:UCSF”>2009 UCSF iGEM team</a>, a paper by <a href=”http://limlab.ucsf.edu/papers/pdfs/park_2014.pdf”>Park et. al.</a>), 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.

Figure 1:

Figure 2a:

Figure 2b:

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.

Sequence and Features