Team:Northwestern/Results/Fluorescence

From 2013.igem.org

(Difference between revisions)
(Created page with "{{:Team:Northwestern/Templates/Skinning}} <html> <style> p { color:black; font-family: Helvetica; text-indent: 30px; } p b { font-family: Helvetica;} .con...")
 
(13 intermediate revisions not shown)
Line 179: Line 179:
       border: solid 1px;
       border: solid 1px;
}
}
 +
 +
</style>  
</style>  
-
<div class = "container">
+
</br>
-
<div class = "menu">
+
-
<ul>
+
-
<li><a href="https://2013.igem.org/Team:Northwestern">Home</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Northwestern/Team">Team</a>
+
-
<ul>
+
-
<li><a href="https://igem.org/Team.cgi?year=2013&team_name=Northwestern">Official Profile</a></li>
+
-
<li><a href="#">Meet Us!</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Northwestern/Attributions">Attribution</a></li>
+
-
</ul>
+
-
</li>
+
-
<li><a href="https://2013.igem.org/Team:Northwestern/Project">Project</a>
+
-
<ul>
+
-
<li><a href="https://2013.igem.org/Team:Northwestern/Parts">Parts Submitted</a></li>
+
-
<li><a href="https://2013.igem.org/Team:Northwestern/Notebook">Notebook</a></li>
+
-
<li><a href="#">Description</a></li>
+
-
</ul>
+
-
</li>
+
-
<li><a href="https://2013.igem.org/Team:Northwestern/Modeling">Modeling</a></li>
+
-
<li><a href= "https://2013.igem.org/Team:Northwestern/Safety"> Safety </a></li>
+
-
</ul>
+
-
</div>
+
-
</div>
+
<div>
<div>
-
<h1> Detection of pH Levels </h1>
+
<h1> Single State Characterization </h1>
 +
 
 +
<h4>Fluorescence Assay </h4>
 +
<p>Due to time constraint, only single state promoters were fully characterized. However, it will be ready for the final presentation. The objective of this assay is to determine the activity of the promoters under a range of pH conditions, ranging from 3.5 to 7.5. Each construct was done in triplicate for each pH.  See the protocol section for a detailed protocol followed. </br></p>
 +
 
 +
<img src = "https://static.igem.org/mediawiki/2013/d/d0/NorthwesternLRG_OD.png" height = "384" width = "672"> </br> </br>
 +
<img src = "https://static.igem.org/mediawiki/2013/5/5e/NorthwesternARG_OD.png" height = "384" width = "672"> </br> </br>
 +
<img src = "https://static.igem.org/mediawiki/2013/3/33/GadA_OD.png" height = "384" width = "672"> </br> </br>
 +
 
 +
 
 +
<p>The plate reader results echoed the results found in the overnight tube tests in that the maximum optical density increases as pH increases. In fact, both pH 3.5 and 4.5 both had negligible growth. Despite the difference in the maximum optical densities, all three constructs, Lpp-RBS-GFP (LRG), GadA-RBS-GFP (GRG), and Asr-RBS-GFP (ARG), all had the same growth rate, independent of the pH. This suggests that the pH had no effect on the bacteria’s ability to grow. Instead, the more acidic conditions force the bacteria to stop growth earlier because it faces pH limited growth.</br></p>
 +
 
 +
<p>The fluorescence of data confirmed the ASR promoter as an acidic promoter. It only activates for pH 5.5 with only basal level of fluorescence for all other pH. However, GadA does not seem to have the same level of pH control. Increasing pH corresponds to increasing fluorescence levels. This characterization presents a different result that the data from the 2010 University of Wisconsin iGEM team. The wider range of pH in this assay also shows a fuller picture of how the promoter behaves.  The Lpp promoter behaves as expected. It has the highest amount of GFP than either of the acidic responsive promoters in all pH ranges. It also shows little signs of any pH responsive element to this constitutive promoter. </br></p>
 +
 
 +
<img src = "https://static.igem.org/mediawiki/2013/e/ec/NorthwesternLRG_Fl.png" height = "384" width = "672"> </br> </br>
 +
<img src = "https://static.igem.org/mediawiki/2013/d/d9/NorthwesternARG_Fl.png" height = "384" width = "672"> </br> </br>
 +
<img src = "https://static.igem.org/mediawiki/2013/6/60/GadA_FL.png" height = "384" width = "672"> </br> </br>
 +
 
 +
<h4>Normalized Data</h4>
 +
 +
<p>Since we are ultimately interested in the promoter activity, the data needs to be normalized to see how much GFP each cell is producing. In addition, due to the limitations of the plate reader, the background noise also needs to be accounted for in the data. To do this, the background media noise, in the form of a non-zero absorbance 600 value and fluorescence value, is subtracted from each data point, and the background-adjusted fluorescence data is then divided by the optical density values to obtain the fluorescence per optical density reading. This value is proportional to the amount of GFP per cell which is ultimately what correlates to the promoter activity. </br></p> </br>
 +
 
 +
<img src = "https://static.igem.org/mediawiki/2013/e/ec/NorthwesternLRG_Fl.png" height = "384" width = "672"> </br> </br>
 +
<img src = "https://static.igem.org/mediawiki/2013/d/d9/NorthwesternARG_Fl.png" height = "384" width = "672"> </br> </br>
 +
<img src = "https://static.igem.org/mediawiki/2013/6/60/GadA_FL.png" height = "384" width = "672"> </br> </br>
-
<h4>Why use a pH-inducible promoter? </h4>
+
<p> The data shown above is missing some pH data systems, such as pH = 3.5. The small numbers for the optical density  is due to a lack of growth caused by great fluctuations that range from extremely high numbers to very negative numbers. If the data are included, they would disrupt one's ability to see the trends. As a result, they are left out. In addition, it is clear from the raw data that the cells did not grow. Therefore, analyzing the fluorescence per optical density for these cells is meaningless. </p>
-
<p> In order to execute the alkalinity response only when it is needed,, it was first necessary to identify pH-inducible promoters that are active at or near pH 5.5 (citation needed). <b>At pH 5.5 or below, the rate of demineralization of the tooth exceeds that of the re-mineralization process provided by saliva (citation needed). </b>This results in erosion of the hard tissues of the tooth (citation needed). <b>Thus, a promoter induced at pH 5.5 provides useful transcriptional control over genes that might prevent the progression of tooth decay.</b><p>
+
-
<h4>Identifying a pH-inducible promoter</h4>
+
<p>The normalized fluorescence data show an interesting result. The general trend for both LRG and GRG is that the the fluorescence per optical density does not differ significantly in different pH range. Since only cells under pH of 5.5, 6.5, and 7.5 grew, the erratic data for cells growing under pH of 3.5 and 4.5 mean very little and can be attributed to the lower limits of the plate reader instrument. While the LRG’s independence of pH is expected, the similarity of GRG’s fluorescence per optical density for pH 5.5 - 7.5 is very surprising. This normalized data is a result of the curves discussed above and further affirm the conclusions made previously. </br> </p>
-
<p>The E. coli genome contains genes which demonstrate elevated levels of transcription at or near pH 5.5. <b>Tucker, et al. (http://jb.asm.org/content/184/23/6551.full) performed a comprehensive study that identified acid-inducible genes contained within the E. coli genome.</b> The team compared the expression levels of genes in cells grown at pH 5.5 to cells grown in pH 7.4. They <b>found that the transcription of the asr and gadA genes were considerably induced at pH 5.5.</b> In fact, the asr gene was the most significantly pH-induced gene identified. <b>Our team proceeded with isolating the promoters of the asr and gadA genes to be used as the pH-inducible promoters driving elevated levels of gene expression within our dual-state promoter.</b></p>
+
-
<h4>GadA is a crucial component in the acid stress response of E. coli</h4>
+
<p>In contrast to GRG, ARG shows a very strong separation for pH 5.5 versus the rest of the pH range. While the normalized fluorescence is relatively low for all other pH, the normalized fluorescence for pH 5.5 is significantly higher, showing that the promoter is indeed turning on at this pH, again affirming the conclusions made above. </br></p>
-
    <p>On the other hand, the gadA gene is better-characterized. The gadA gene is a part of the gad system, which is an acid-inducible glutamate decarboxylase-based acid resistance system that enables the survival of E. coli under acid stress conditions. (http://jb.asm.org/content/188/23/8118.full).  <b>GadA is a gene encoding a glutamate decarboxylase.</b> (http://jb.asm.org/content/185/15/4644.full,  http://jb.asm.org/content/188/23/8118.full)<b> The process of decarboxylating glutamate consumes protons that leach into the cell under acid stress. </b>(http://jb.asm.org/content/188/23/8118.full)<b>  In this manner, the gad system manages protons that would otherwise drop the cellular pH below levels at which E. coli could survive.</b></p>
+
-
    <p>The function of the gad system explains why these genes experience high transcription levels at low pH. Despite this functional explanation, modes of transcriptional control over the gad system are rather complex. Transcriptional factors RpoS, cyclic AMP receptor protein, HN-S and EvgA all play a role in transcriptional regulation. INCOMPLETE SECTION -Do more research, since this section is complicated. </p>
+
-
<h4>Identification of gadA and asr promoter regions</h4>
+
<h4> Conclusion?? </h4>
-
<h4>Transcriptional regulation of asr promoter</h4>
 
-
      <p><b>As of yet, the function of the asr gene, or “acid-shock RNA” gene, and the mechanism responsible for its induction are still unclear.</b> However, Iien et al. have taken significant steps toward characterizing the gene. They propose that<b> asr encodes a periplasmic or outer-membrane protein.</b> Knockout experiments illustrated that the PhoBR operon plays a significant role in activating the asr gene. They demonstrated through mobility shift electrophoresis that the PhoB protein binds to the promoter region of asr.  By analyzing the sequence of the asr promoter region, they revealed that it contains a sequence similar to that of the Pho box, which is a consensus sequence known to bind the PhoB protein. The Pho box can be found in the promoter regions of other PhoB-regulated genes. (http://jb.asm.org/content/181/7/2084.long)This evidence suggests that<b>the regulatory protein PhoB indeed exerts some transcriptional control over the asr gene. </b></p>
 

Latest revision as of 03:30, 28 September 2013


Single State Characterization

Fluorescence Assay

Due to time constraint, only single state promoters were fully characterized. However, it will be ready for the final presentation. The objective of this assay is to determine the activity of the promoters under a range of pH conditions, ranging from 3.5 to 7.5. Each construct was done in triplicate for each pH. See the protocol section for a detailed protocol followed.







The plate reader results echoed the results found in the overnight tube tests in that the maximum optical density increases as pH increases. In fact, both pH 3.5 and 4.5 both had negligible growth. Despite the difference in the maximum optical densities, all three constructs, Lpp-RBS-GFP (LRG), GadA-RBS-GFP (GRG), and Asr-RBS-GFP (ARG), all had the same growth rate, independent of the pH. This suggests that the pH had no effect on the bacteria’s ability to grow. Instead, the more acidic conditions force the bacteria to stop growth earlier because it faces pH limited growth.

The fluorescence of data confirmed the ASR promoter as an acidic promoter. It only activates for pH 5.5 with only basal level of fluorescence for all other pH. However, GadA does not seem to have the same level of pH control. Increasing pH corresponds to increasing fluorescence levels. This characterization presents a different result that the data from the 2010 University of Wisconsin iGEM team. The wider range of pH in this assay also shows a fuller picture of how the promoter behaves. The Lpp promoter behaves as expected. It has the highest amount of GFP than either of the acidic responsive promoters in all pH ranges. It also shows little signs of any pH responsive element to this constitutive promoter.







Normalized Data

Since we are ultimately interested in the promoter activity, the data needs to be normalized to see how much GFP each cell is producing. In addition, due to the limitations of the plate reader, the background noise also needs to be accounted for in the data. To do this, the background media noise, in the form of a non-zero absorbance 600 value and fluorescence value, is subtracted from each data point, and the background-adjusted fluorescence data is then divided by the optical density values to obtain the fluorescence per optical density reading. This value is proportional to the amount of GFP per cell which is ultimately what correlates to the promoter activity.








The data shown above is missing some pH data systems, such as pH = 3.5. The small numbers for the optical density is due to a lack of growth caused by great fluctuations that range from extremely high numbers to very negative numbers. If the data are included, they would disrupt one's ability to see the trends. As a result, they are left out. In addition, it is clear from the raw data that the cells did not grow. Therefore, analyzing the fluorescence per optical density for these cells is meaningless.

The normalized fluorescence data show an interesting result. The general trend for both LRG and GRG is that the the fluorescence per optical density does not differ significantly in different pH range. Since only cells under pH of 5.5, 6.5, and 7.5 grew, the erratic data for cells growing under pH of 3.5 and 4.5 mean very little and can be attributed to the lower limits of the plate reader instrument. While the LRG’s independence of pH is expected, the similarity of GRG’s fluorescence per optical density for pH 5.5 - 7.5 is very surprising. This normalized data is a result of the curves discussed above and further affirm the conclusions made previously.

In contrast to GRG, ARG shows a very strong separation for pH 5.5 versus the rest of the pH range. While the normalized fluorescence is relatively low for all other pH, the normalized fluorescence for pH 5.5 is significantly higher, showing that the promoter is indeed turning on at this pH, again affirming the conclusions made above.

Conclusion??