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. Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 643
Illegal XhoI site found at 1042 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 777
Illegal AgeI site found at 151 - 1000COMPATIBLE WITH RFC[1000]
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 comparing to the CMV promoter, and these levels are almost equal between the WT and the mutant promoter.
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