Team:MIT/Results/

From 2011.igem.org

(Difference between revisions)
Line 70: Line 70:
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.
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.
-
<p>Hek293 results here
+
<4>Hek293 Transfection Results:
-
<p><img src="https://static.igem.org/mediawiki/2011/0/0d/Specimen_001_Charles_2.fcs_scatter.jpg" style="max-width:400px; margin-right:10px;"/><img src="https://static.igem.org/mediawiki/2011/0/0d/Specimen_001_Charles_2.fcs_scatter.jpg" style="max-width:400px; margin-right:10px;"/>
+
<p><img src="https://static.igem.org/mediawiki/2011/0/0d/Specimen_001_Charles_2.fcs_scatter.jpg" style="max-width:400px; margin-right:10px;"/><img src="https://static.igem.org/mediawiki/2011/f/fa/Specimen_001_Charles_2.fcs.jpg" style="max-width:400px; margin-right:10px;"/>
 +
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).
 +
<p>Although each

Revision as of 21:36, 26 September 2011

Navigation

  • DNA Delivery
  • Parts Constructed
  • Parts Characterizations
  • Patterning Modeling Results
  • Patterning Experimental Results
  • Cell Adhesion Results
  • Attributions

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. <4>Hek293 Transfection Results:

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

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

Name: Hef1a-LacOid MammoBlock ID: BBa_K511000
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.

Description/Usage:
Characterization Link
ADD MORE PARTS WITH SAME FORMAT

Parts Characterizations

The characterization of newly constructed biological parts is ADD TO BLURB

List of characterizations

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 here

Patterning 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.