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Revision as of 21:28, 26 September 2011
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 results here
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
Type: Regulatory Length: 1275
This part encodes a promoter with low-level, constitutive activity that can be repressed by variants of the LacI transcriptional repressor. Repression by LacI-KRAB through chromatin packing is quite effective.
Parts Characterizations
The characterization of newly constructed biological parts is ADD TO BLURB
List of characterizations
- rtTA3/TRE promoter
- Gal4/UAS promoter
- LacI/Hef1a-LacO repressor
- Delta-Notch interaction
- CMV-TetO promoter
- Mnt-VP16/Mnt promoter
- CI434-VP16/CI434 promoter
- Gal4→LacI¬rtTA3→Reporter cascade (!)
rtTA3/TRE promoter
Explanation:
EXPLAIN HERE
Gal4/UAS promoter
Explanation:
EXPLAIN HERE
LacI/Hef1a-LacO repressor
Explanation:
EXPLAIN HERE
Delta-Notch interaction
Explanation:
EXPLAIN HERE
CMV-TetO promoter
Explanation:
EXPLAIN HERE
Mnt-VP16/Mnt promoter
Explanation:
EXPLAIN HERE
CI434-VP16/CI434 promoter
Explanation:
EXPLAIN HERE
Gal4→LacI¬rtTA3→Reporter cascade
Explanation:
EXPLAIN HERE
Patterning Modeling Results
Semon's Modeling Results goes herePatterning Experimental Results
Here we show the results that we have obtained thus far in using our Notch-Delta system. We have tried various combinations of Hek and CHO cell co-cultures to show that the Notch-Delta system is in fact functional.Cell Adhesion Experimental Results
Cadherins are an important Calcium-dependent surface protein that can bind to same-type cadherins on other cells. The existence of different types of cadherins allows for the the development of highly organized tissues, as the sequential expression of various types of cadherins in various cells drives the development of complex three-dimensional structures.With this in mind, we brought cadherins into our project with the goal of forming self-adhesive patterns when tied into the Notch-Delta and internal logic processing systems. Below we have some initial results.
Attributions
Our instructors were very helpful not only in giving feedback on our designs, cloning strategies, and data, but also in training us for lab work. The training for tissue culture work was conducted by Linda Stockdale in the Griffiths lab. Gibson assembly techniques and FACS training from Deepak Mishra, one of our instructors.
Other than the initial training, all work was done by our undergraduate team.
Special credit belongs to Semon Rezchikov and Jenny Cheng for simulations and modeling, and Tiffany Huang for wiki design.