Part:BBa_K2644210
pEGFP-N3
This is the DNA sequence of GFP protein.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Characterization
This part shares general concepts with the Valencia 2012 part: BBa_K1123005
Usage
BBa_K2644210 is a basic part containing coding sequence of EGFP protein. When using this part, you should cut the coding sequence down and connect it with a plasmid which is mainly used to express proteins. And after transfected the assembled plasmid successfully into the cells, the eGFP gene will be translated to eGFP protein at last and therefore the cells express eGFP steadily can be observed with green fluorescent.
Biology
EGFP is an enhanced green fluorescent protein, a mutant of GFP protein. At present, eGFP is widely used since it emits more than 6 times of fluorescence intensity than GFP, thus it is more suitable as a reporter than GFP, and is a better choice to show gene expression, regulation, cell differentiation, protein localization and transport in cells. In our experient, we are expected to characterize the cutting ability of sgRNA/Cas9 by fluorescent in cells.
Results
We firstly amplified the eGFP fragment from BBa_K1123005 (iGEM13_TU-Eindhoven) by PCR.
Figure 1. Amplification of eGFP fragment (BBa_K763002) by PCR. Lane M, marker. Lane 1, 25ng eGFP fragment. Lane 2, 50ng. Lane 3, 100ng. Lane 4, 150ng. Lane 5, 200ng.
Since this fragment contains the cleavage site of our sgRNA, we used it to test the in vitro cleavage activity of our CRISPR/Cas9 system. Please notice that our cleavage site was not so suitable for this fragment since it was designed for plasmid cutting(fragment of 720bp is cut into fragments of about 80bp and 640bp).
Figure 2. In vitro cleavage of eGFP fragment. Lane M, marker. Lane 1, eGFP fragment. Lane 2-3, sgRNA:Cas9:DNA=10:10:1. Lane 4-5, sgRNA:Cas9:DNA=10:20:1. Lane 6-7, sgRNA:Cas9:DNA=20:20:1.
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