Team:MIT/Results/

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<p>(1A) This 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 were constitutively active, expressing yellow and 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 in mKate fluorescence (PE-TexasRed channel). (1B) Histogram of signal intensities of the above data. (1C) The negative control sample was transfected with plasmids containing no functional promotor-gene pairs.
<p>(1A) This 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 were constitutively active, expressing yellow and 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 in mKate fluorescence (PE-TexasRed channel). (1B) Histogram of signal intensities of the above data. (1C) The negative control sample was transfected with plasmids containing no functional promotor-gene pairs.
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<p>We were then interested in the efficiency of multiple plasmid transfections. Previous work suggests that the probabilities of uptaking different plasmids is not independent. Cells that have been succesfully transfected with one plasmid are more likely to also have been transfected with another. 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 <b>99.8%</b> 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.
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<p>We were then interested in the efficiency of multiple plasmid transfections. Previous work suggests that the probabilities of uptaking different plasmids is not independent. Cells that have been succesfully transfected with one plasmid are more likely to also have been transfected with another. To show this correlation, we decided to see what percentage of cells that took up the Hef1a_mKate plasmid also took up the Hef1a-LacO_eYFP plasmid. In order to evaluate the correlation, we took all events above the observed basal PE-TexasRed fluorescence level and examined their yellow fluorescence profile. We observe in the below graph that <b>more than 99%</b> of the red population display yellow fluorescence above basal levels. We make use of this high level of correlation later.
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<p><img src="https://static.igem.org/mediawiki/2011/7/7e/Specimen_001_Charles_2.fcs_scatter_gated.jpg" style="max-width:550px; margin-right:10px;"/>
<p><img src="https://static.igem.org/mediawiki/2011/7/7e/Specimen_001_Charles_2.fcs_scatter_gated.jpg" style="max-width:550px; margin-right:10px;"/>
<img src="https://static.igem.org/mediawiki/2011/1/1c/Specimen_001_Charles_2.fcs_gated.jpg" style="max-width:550px; margin-right:10px;"/>
<img src="https://static.igem.org/mediawiki/2011/1/1c/Specimen_001_Charles_2.fcs_gated.jpg" style="max-width:550px; margin-right:10px;"/>
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<p>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.
<p>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.

Revision as of 21:03, 27 September 2011

Navigation

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

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 from Invitrogen, and obtained at best an 80% transfection efficiency for Hek293 cells and 10% transfection efficiency for CHO cells.

Figure 1: Hek293 Lipofectamine Transfection Results

A

B


C


(1A) This 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 were constitutively active, expressing yellow and 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 in mKate fluorescence (PE-TexasRed channel). (1B) Histogram of signal intensities of the above data. (1C) The negative control sample was transfected with plasmids containing no functional promotor-gene pairs.

We were then interested in the efficiency of multiple plasmid transfections. Previous work suggests that the probabilities of uptaking different plasmids is not independent. Cells that have been succesfully transfected with one plasmid are more likely to also have been transfected with another. To show this correlation, we decided to see what percentage of cells that took up the Hef1a_mKate plasmid also took up the Hef1a-LacO_eYFP plasmid. In order to evaluate the correlation, we took all events above the observed basal PE-TexasRed fluorescence level and examined their yellow fluorescence profile. We observe in the below graph that more than 99% of the red population display yellow fluorescence above basal levels. We make use of this high level of correlation later.

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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 Lipofectamine Transfection Results

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

Our Favorite Parts

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
Name: UAS-Gal4 Promoter MammoBlock ID: BBa_K511003
Type: Regulatory Length: 220

This part encodes a promoter that is inducible by variants of the Gal4 transactivator and off otherwise.

Description/Usage:
Characterization Link
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

Other Parts We Like

Parts Characterizations

The characterization of newly constructed biological parts is ADD TO BLURB

List of characterizations

Delta-Notch interaction

Explanation:

EXPLAIN HERE

rtTA3/TRE promoter

Explanation:

EXPLAIN HERE

Gal4/UAS promoter

Explanation:

EXPLAIN HERE

LacI/Hef1a-LacO repressor

Explanation:

EXPLAIN HERE

Gal4-VP16→LacI¬rtTA3→Reporter cascade

Explanation:

Here we show an experimental result of an example of what we call our internal processing module, which is essentially a cascade of several transcription factors and their corresponding binding sites. Taking the individual functional transcription factor systems characterized above, we combine them into a cascade to allow for higher-level control. Our system cascades can be any combination of the promoters UAS (which is activated by Gal4-VP16), TRE (activated by rtTA3 in the presence of doxycycline), and Hef1a-LacO (repressed by LacI in the absence of IPTG). In this particular experiment, we used the following genetic construct: Hef1a_Gal4-VP16, UAS_LacI, Hef1a-LacO_rtTA3, TRE_Delta-mCherry. The constitutive Hef1a_Gal4-VP16 is intended to mimic the signal that would come from an activated Notch protein, and the final repression of our Delta-mCherry protein through repression of the Hef1a-LacO_rtTA3 intermediate part mimics the cellular response of turning off Delta production. The concentration of IPTG and rtTA3 present would then serve as an external control of the extent to which Delta production is reduced.

The above result, obtained from flow cytometry by measuring the mCherry fluorescence of transfected Hek293 cells in the presence of different IPTG and doxycycline concentrations, are as we expected. In the absence of doxycycline, rtTA3 cannot activated the TRE promoter, and with increasing IPTG concentration, expressed LacI cannot inhibit the Hef1a-LacO promoter, and so we see a rise in Delta-mCherry due to this.

CMV-TetO promoter

Explanation:

EXPLAIN HERE

Mnt-VP16/Mnt promoter

Explanation:

EXPLAIN HERE

CI434-VP16/CI434 promoter

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 being able to directly control cadherins in mammalian cells, leading to the formation of self-adhesive patterns when tied into the Notch-Delta and internal logic processing systems. Below we have some initial results.

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 for simulations and modeling, Jenny Cheng for animations, and Tiffany Huang for wiki design.