DNA Delivery Systems

Transfection Using Lipofectamine 2000 (Invitrogen)

To introduce our engineered genetic parts into mammalian cells, we employed the Lipofectamine 2000 reagent, and obtained at best an 80% transfection efficiency for Hek293 cells and 10% transfection efficiency for CHO cells.

Hek293 Transfection Results:

Control image:

This FACS scatter plot shows the distribution of the Hek293 population after it was transfected with the following DNA parts: Hef1a-LacO_eYFP and Hef1a_mKate, both of which are constitutive promoters in the absence of LacI driving eYFP (yellow) and mKate (red) fluorescent proteins, respectively. We observe a distinct shift of approximately 83% of the population in their eYFP fluorescence (FITC channel), while we observe a 69% shift mKate fluorescence (PE-TexasRed channel).

Although each of our constructed DNA parts contains Gibson sequences flanking the promoter and gene of interest, we have not yet used the Gibson reaction to piece together two or more promoter-gene pairs. The construction involved in doing so would involve the generation of a large library of promoter-gene pairs with various Gibson sequences that would be compatible with those of other parts that we intend to experiment with. In short, we have not yet engaged in Gibson-reacting parts together, and instead preserve our experimental flexibility through co-transfection of multiple plasmids.

CHO results here

Transfection By Nucleofection

Although Hek293 cells are very easy to transfect and are therefore a very suitable target for demonstration, we found during the summer that cadherins are endogenously expressed, and this limits their experimental flexibility when it comes to cell-cell adhesion. CHO cells, however, do not have the same problem. Seeing also that the Notch-Delta system was previously characterized by the Elowitz group using CHO cells, we decided to open up a parallel research channel with CHO cell transfections. As documented above, however, lipofectamine proved to be an exceedingly difficult and somehow unsuccessful method of transfection into CHO cells, so we thus moved to nucleofection.

Parts Constructed

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