Part:BBa_K2520023

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 (2016). 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: GFP expression under CMV, EF1a WT and mutant EF1a promoters.

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

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 643
  Illegal XhoI site found at 1042
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 777
  Illegal AgeI site found at 151
  
• 1000
  COMPATIBLE WITH RFC[1000]