Team:MIT/Treatment

From 2014.igem.org

(Difference between revisions)

Latest revision as of 03:59, 18 October 2014

Description
Experiments

BACE1
BACE2

Parts

TREATMENT MODULE

SUBGROUP MEMBERS: James T Anderson, Alex Laffell

Attributions: James T Anderson

Down-regulating Aβ production and up-regulating Aβ degradation

Free JavaScripts provided
by The JavaScript Source

Each of our three planned experiments for determining our effectiveness at re-regulating BACE1/BACE2 expression relied on successful transfection of our vectors into HEK293. Unfortunately, continual struggles with low transfection efficiency precluded us from performing all but our FRET experiment. From this experiment we dected less Bace1 activity in the four samples transfected with each of our four miRNA's.

Description

The Treatment module is actuated upon release of transcription factor rtTA, initiating a two-part response. The first of these two responses is transcriptional up-regulation of an exogenously delivered vector expressing the Bace2 protease. Bace2 recognizes cleavage sites within Aß and so is thought to be a potentially effective therapeutic for degrading plaques.

The second response activated by the release of rtTA is transcriptional expression of a miRNA targeted specifically to BACE1. We constructed four independent miRNA-generating vectors, each targeted to a different region of the BACE1 mRNA sequence. The benefit of having multiple unique miRNA’s is the ability to test the relative efficacy of each at down-regulating BACE1 translational expression. Predicting an effective miRNA depends on a variety of factors ranging from the sequence of bases that flank the guide region, to the degree of complementarity between the miRNA and its target, to the region of the target that the miRNA binds.

return to top

Experiments

return to top

Transfection for BACE1 Experiments

To test our miRNA’s effectiveness at down-regulating BACE1 expression we transfected separate HEK293 cultures with either (1) BACE1 under constitutive expression by the Hef1a promoter or (2) with BACE1 under constitutive expression by the Hef1a promoter and with a miRNA-generating vector inducibly regulated by the TRE promoter. We performed these transfections in duplicate—once with native BACE1, and then once with eYFP-tagged BACE1. There were two variants of this eYFP-tagged BACE1 construct—one with eYFP linked to the N-terminus of Bace1 and another with eYFP on the C-terminus. We created these two variants after considering the possibility that linking eYFP to either terminus of Bace1 might interfere with the peptide’s native structure or function.

After transfection and Doxycycline-induction of our miRNA-generating vectors, we used flow cytometry to verify that the miRNA-generating vectors were being expressed. Proper splicing of the mature miRNA out of the expression vector leaves an intact mKate coding sequence in the vector, so emission of red fluorescence from our transfected HEK293 indicates proper miRNA processing.

miRNA1

miRNA2

miRNA3

miRNA4

Fig. 1. Determining whether miRNA's 1-4 are successfully processed. The data represent transfection with Hef1a:BACE1, Hef1a:tagBFP, and each of the listed miRNA's. Expression of each miRNA was induced by treatment with 1uM Doxycycline.

return to top

Transfection for BACE2 Experiments

The one important difference between the BACE1 and BACE2 experiments was only that the BACE2 experiment required a simpler initial transfection. We transfected two cultures of HEK293 with either (1) a vector inducibly expressing BACE2 under control of the TRE promoter or (2) with a vector inducibly expressing BACE2 under control of a TRE promoter and with a vector constitutively expressing the rtTA transcription factor. So by comparing the amount of BACE2 expression in the induced versus un-induced transfection samples, we could determine our effectiveness at up-regulating BACE2 expression.

Following confirmation of successful transfection via flow cytometry, we planned three experiments to assay the change in BACE1/2 expression.

First Experiment: FRET Assay

The first experiment was based on Anaspec’s SensoLyte 520 β-Secretase Assay Kit. This commercial kit uses a FRET-based technique wherein biological samples are mixed with a proprietary solution containing a BACE1/2 mimic substrate. One terminal of this mimic substrate is conjugated to a fluorophore and the other terminal is conjugated to a fluorescence quencher. So proteolytic Bace1/2 activity causes cleavage of the proprietary substrate, which increases fluorescence due to separation of the fluorophore from the quencher. Relative Bace1 activity in biological samples can be assayed by measuring the intensity of the fluorophore emission and comparing to a standard curve.

Fig.2. Determining miRNA efficacy in down-regulating BACE1 expression. 30,000,000 cells from each sample were analyzed. RFU represents "relative fluorescence units", a proxy for determining Bace1 protease activity. The shown fluorescence values represent RFU after background fluorescence (as determinined by substrate-only control samples) was subtracted from raw output.

Second Experiment: Detecting Fluorescence from YFP-tagged BACE1/2

The second planned experiment involved the previously mentioned eYFP-tagged Bace1/2 construct. Comparison of yellow fluorescence in miRNA-transfected versus miRNA-untransfected HEK293 would offer evidence as to whether BACE1/2 expression increases or decreases under the regulation of our miRNA’s. Furthermore, microscopy would allow us to visualize where Bace1/2 localizes in the cell.

Third Experiment: RT-PCR

Our third intended experiment was RT-PCR. We planned to isolate bulk RNA from the transfected HEK293, perform cDNA synthesis on the isolated RNA, and then use quantitative RT-PCR to determine the relative amount of Bace1/2 mRNA in miRNA-transfected versus miRNA-untransfected HEK293 to determine the efficacy of our miRNA’s in targeting BACE1/2 mRNA for degradation.

return to top

Parts

full parts list available here
• TRE: miRNA1
• TRE: miRNA2
• TRE: miRNA3
• TRE: miRNA4
• Hef1a: BACE1
• Hef1a: eYFP-BACE1
• Hef1a: BACE1-eYFP
• TRE: BACE2
• TRE: eYFP-BACE2
• TRE: BACE2-eYFP

Retrieved from "http://2014.igem.org/Team:MIT/Treatment"

@@ Line 22: / Line 22: @@
 <center>
 <p style="font-size:12px" align=center><i><b>SUBGROUP MEMBERS: James T Anderson, Alex Laffell</b></i></p>
-<p style="font-size:12px" align=center><i>Attributions: James T Anderson (???)</i></p>
+<p style="font-size:12px" align=center><i>Attributions: James T Anderson</i></p>
 <font size=4><br />Down-regulating Aβ production and up-regulating Aβ degradation</font><br /><br />
@@ Line 58: / Line 58: @@
 </td></tr></table>
-<center>Each of our three planned experiments for determining our effectiveness at re-regulating BACE1/BACE2 expression relied on successful transfection of our vectors into HEK293. Unfortunately, continual struggles with low transfection efficiency precluded us from performing any of our intended experiments. Analysis of our transfected HEK293 cultures with flow cytometry revealed very low numbers of live cells, suggesting that something had gone awry in the transfection protocol, in the quality of our vector constructs, or in our FACS preparation process. In any case, the lack of usable HEK293 meant we had no samples on which to perform any of our three intended experiments. This is an issue we hope to resolve soon so that we can make meaningful conclusions about the success of the Treatment module.<br /></center>
+<center>Each of our three planned experiments for determining our effectiveness at re-regulating BACE1/BACE2 expression relied on successful transfection of our vectors into HEK293. Unfortunately, continual struggles with low transfection efficiency precluded us from performing all but our FRET experiment. From this experiment we dected less Bace1 activity in the four samples transfected with each of our four miRNA's.<br /></center>
 <h2>Description</h2><a name="description"></a>
@@ Line 94: / Line 94: @@
 </td>
 <tr><td width="100%">
-<p> <b>Determining whether miRNA's 1-4 are successfully processed.</b><i> The data represent transfection with Hef1a:BACE1, Hef1a:tagBFP, and each of the listed miRNA's. Expression of each miRNA was induced by treatment with 1uM Doxycycline. </i></p></td>
+<p> <b>Fig. 1. Determining whether miRNA's 1-4 are successfully processed.</b><i> The data represent transfection with Hef1a:BACE1, Hef1a:tagBFP, and each of the listed miRNA's. Expression of each miRNA was induced by treatment with 1uM Doxycycline. </i></p></td>
 </tr>
 </table><br><a href="#top">return to top</a>
@@ Line 100: / Line 100: @@
 The one important difference between the BACE1 and BACE2 experiments was only that the BACE2 experiment required a simpler initial transfection. We transfected two cultures of HEK293 with either (1) a vector inducibly expressing BACE2 under control of the TRE promoter or (2) with a vector inducibly expressing BACE2 under control of a TRE promoter and with a vector constitutively expressing the rtTA transcription factor. So by comparing the amount of BACE2 expression in the induced versus un-induced transfection samples, we could determine our effectiveness at up-regulating BACE2 expression. <br /> <br />
-Following confirmation of successful transfection via flow cytometry, we planned three experiments to assay the change in BACE1 expression by miRNA’s. <br /> <br />
+Following confirmation of successful transfection via flow cytometry, we planned three experiments to assay the change in BACE1/2 expression. <br /> <br />
 <b>First Experiment: FRET Assay</b><br /> <br />The first experiment was based on Anaspec’s SensoLyte 520 β-Secretase Assay Kit. This commercial kit uses a FRET-based technique wherein biological samples are mixed with a proprietary solution containing a BACE1/2 mimic substrate. One terminal of this mimic substrate is conjugated to a fluorophore and the other terminal is conjugated to a fluorescence quencher. So proteolytic Bace1/2 activity causes cleavage of the proprietary substrate, which increases fluorescence due to separation of the fluorophore from the quencher. Relative Bace1 activity in biological samples can be assayed by measuring the intensity of the fluorophore emission and comparing to a standard curve. <br /> <br />
-<img src="https://static.igem.org/mediawiki/2014/4/49/Bace1_anaspec.png" width=2000><br><br>
+<img src="https://static.igem.org/mediawiki/2014/4/49/Bace1_anaspec.png" width=600px><br><br>
+<b>Fig.2. Determining miRNA efficacy in down-regulating BACE1 expression.</b><i> 30,000,000 cells from each sample were analyzed. RFU represents "relative fluorescence units", a proxy for determining Bace1 protease activity. The shown fluorescence values represent RFU after background fluorescence (as determinined by substrate-only control samples) was subtracted from raw output. </i><br/><br/>
 <b>Second Experiment: Detecting Fluorescence from YFP-tagged BACE1/2</b><br /> <br />The second planned experiment involved the previously mentioned eYFP-tagged Bace1/2 construct. Comparison of yellow fluorescence in miRNA-transfected versus miRNA-untransfected HEK293 would offer evidence as to whether BACE1/2 expression increases or decreases under the regulation of our miRNA’s. Furthermore, microscopy would allow us to visualize where Bace1/2 localizes in the cell. <br /> <br />