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Team:Cambridge-JIC/Marchantia/EnhancerTrap
From 2014.igem.org
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<h3>To sum up the biology of the construct:</h3> | <h3>To sum up the biology of the construct:</h3> | ||
<ol> | <ol> | ||
| - | <li color="red">A red fluorescent protein expressed all the time, everywhere (to check construct insertion)</li> | + | <li><font color="red">A red fluorescent protein expressed all the time, everywhere (to check construct insertion)</font></li> |
<li>A hygromycin selectable marker (to allow only transformant to survive)</li> | <li>A hygromycin selectable marker (to allow only transformant to survive)</li> | ||
<li>A minimal promoter fused to a gal4 sequence (gal4 will be expressed if minimal prom has been inserted next to an enhancer)</li> | <li>A minimal promoter fused to a gal4 sequence (gal4 will be expressed if minimal prom has been inserted next to an enhancer)</li> | ||
Revision as of 13:00, 8 October 2014
How does an Enhancer Trap work?
An enhancer trap constructs contains a transcription activator (e.g.: GAL4) fused to a weak or basal promoter (TATA box). The construct also contains a reporter gene (e.g.: YFP) fused to a UAS (upstream activator sequence activated by GLA4). This allows YFP to report the GAL4 expression. On its own the basal promoter is insufficient to drive detectable expression of the reporter gene, but can respond to transcriptional enhancers from the genome it is inserted next to.. Once a transgenic line is generated using such a construct it can be crossed with any other transgenic line containing a UAS::GOI (gene of interest). The offspring will express the gene of interest with the same characteristic seen as the reporter gene (e.g.: YFP).
To sum up the biology of the construct:
- A red fluorescent protein expressed all the time, everywhere (to check construct insertion)
- A hygromycin selectable marker (to allow only transformant to survive)
- A minimal promoter fused to a gal4 sequence (gal4 will be expressed if minimal prom has been inserted next to an enhancer)
- A gal4 induced YFP (to be able to detect expression pattern of gal4 by the ‘trapped enhancer’ )
Construct Design
The construct was formed from two plasmids A and B that were given to us by Jim Haseloff and Bernado Pollak respectively. The minimal promoter was extracted from plasmid A and inserted in plasmid B. The position of the hygromycin cassette and minimal promotergal4 cassette were exchanged to the position seen below. This was done to place the minimal promoter at the very border of the sequence allowing genomic enhancers to act directly on it.
