DNA Delivery Systems and Data Collection

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 flow cytometry 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). In the control sample of Hek293 cells, we transfected an equivalent mass of DNA that did not contain any promoter-gene pairs to serve as the dummy control for our transfections. From the flow cytometry results of our control sample, we can understand the basal fluorescence of the cell type and perform necessary gating operations to better analyze our data.

From a chemical standpoint, it is reasonable to hypothesize that cells which have taken up a certain plasmid will also take up another plasmid present in the mix with a high correlation, as the lipofectamine packages DNA indiscriminately and cells that have been transfected should possess all plasmids packed by lipofectamine. To show this correlation, we decided to see what percentage of cells that took up the Hef1a_mKate plasmid (expressing red) also took up the Hef1a-LacO_eYFP plasmid (expressing yellow). We performed a PE-TexasRed channel gate above the basal fluorescence level of our control cells and observe in the below graph that there is a 99.8% correlation in co-transfection. This high correlation allowed us to confidently use co-transfection as a temporary means of experimentation, although we seek to eventually create and perform transfection of a single plasmid containing all parts of our system. The difficulty and inconvenience of such is described below.

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

Control CHO cells:

In the flow cytometry scatter plot and histogram above we see the fluorescence distribution of a population of CHO cells transfected with Lipofectamine 2000 and Hef1a_eBFP2, a constitutive blue color. We note that CHOs naturally emit autofluorescence, as seen in the small tail pointing upwards in the DAPI (blue) channel. This tail accounts for 8% of the population, and after transfection, only 14.5% exhibit blue fluorescence beyond the majority of the CHO population, giving us thus approximately 6.5% transfection efficiency.

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

ADD MORE PARTS WITH SAME FORMAT