Usage and Biology
This is a part that we have improved from part BBa_S03452, which is a part that contains a EGFP(Enhance Green Fluorescent Protein).
GFP and EGFP are prone to noncovalent dimerization, which can lead to problems such as dimerizing protein of interest or disturbing the homogeneity of protein concentration. To avoid problems brought forward by dimerization, we improved part BBa_S03452 by point-mutating EGFP to changed the Van-Der-Waals force between identical EGFP molecules and decreased EGFP’s propensity to form dimers. This results in a monomer EGFP (mEGFP) which can emit green fluorescence without forming dimers.
Characterization of the part:
1.Length confirmation: Our DNA segment includes the mEGFP sequence and a prefix and suffix, resulting in total length around 778bp.
2.Modelling: We used SWISS-MODEL, an online protein modelling system, to check if our protein likely forms a monomer. We compared the sequence of mEGFP to the original EGFP. The modelling result shows promising predictions.
4.DNA Sequencing Sequencing results has proved our mutation succesful.
3.Functionality Test To make sure that the mEGFP emits green fluorescence, we transfected HEK 293T cells containing mEGFP gene under CMV promoter. As shown in Figure below, cells that are transfected can emit green fluorescent light.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BamHI site found at 724
- 23COMPATIBLE WITH RFC
- 25COMPATIBLE WITH RFC
- 1000COMPATIBLE WITH RFC