Difference between revisions of "Part:BBa K2520023"
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===Results===
 
===Results===
 
In order to test our mutant promoter we created, we tested the expression of the reporter gene GFP under three different promoters- CMV, EF1a native 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 native and the mutant promoter.
 
In order to test our mutant promoter we created, we tested the expression of the reporter gene GFP under three different promoters- CMV, EF1a native 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 native and the mutant promoter.
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[[File:EF1a results.png|800px|thumb|center|Figure 1: GFP expression under CMV, native EF1a and mutant EF1a promoters. ]]
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[[File:EF1a results.jpeg|800px|thumb|center|Figure 1: GFP expression under CMV, native EF1a and mutant EF1a promoters. ]]
  
 
<partinfo>BBa_K2520008 parameters</partinfo>
 
<partinfo>BBa_K2520008 parameters</partinfo>
 
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Revision as of 13:55, 26 October 2017


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 614
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 679
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 2264


Results

In order to test our mutant promoter we created, we tested the expression of the reporter gene GFP under three different promoters- CMV, EF1a native 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 native and the mutant promoter.

Figure 1: GFP expression under CMV, native EF1a and mutant EF1a promoters.