Difference between revisions of "Part:BBa K921002"

Revision as of 15:32, 2 October 2012

T7 RNAP + IPTG->PoPs (Mutant III)
This is a mutant T7 promoter that includes a lacO operator site. The promoter is inhibited by the LacI protein and can be induced by IPTG. The promoter sits in the 5' region of a gene and initiates transcription when cells express T7 RNA polymerase. Please use cell strains that are compatible with T7 expression vectors.
The following parameters were calculated and compared against the "wild-type" promoter.

Promoter	BBa K921000	BBa K921001	BBa K921002
Transcription Strength	97%	72%	127%
Translational Efficiency	6%	6%	94%

Cells were grown in M9 media without casamino acids. Fluorescence intensities were normalized to optical density (absorbance @600nm).

Fluorogen-activating proteins were used as protein reporters in our characterization experiment (see details in here). 10uM malachite green was added to each well of a 96-well plate and fluorescence intensities were recorded over time. Our model was used to simulate the time lapse data using differential equations.

The Spinach aptamer was used as a RNA reporter in our characterization experiment (see details in here). 200uM DFHBI was added to each well of a 96- well plate and fluorescence intensities were recorded over time. Our model was used to simulate the time lapse data using differential equations.

Note: To learn more about how we characterized this set of promoters, click here.

Discussion:
The design of this mutant T7Lac promoter (BBa_K921002) was based on a different class of T7 promoters, which are weaker than the wildtype T7Lac promoter (BBa_K613007). Therefore, this promoter is expected to produce less protein than the wildtype promoter. However, this mutant promoter produces more protein than the wildtype promoter in our experiments. We hypothesize that this promoter does not burden cells as much as the wildtype T7 promoters, hence giving rise to higher translation rate of mRNA.

We note that multiple dips were recorded in the readings of all three mutated promoters. This could be due to a strong metabolic burden and multiphasic growth of bacteria.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
COMPATIBLE WITH RFC[21]
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Functional Parameters

direction	Forward
efficiency	160%
negative_regulators	LacI (Hypothesized LacIQ compatible)

@@ Line 3: / Line 3: @@
 <html>
 <br>
-This is a mutant T7 promoter that includes a lacO sequence. This is repressible by the LacI protein and is induced in the presence of IPTG. The promoter sits in the 5' region of the gene of interest and initiates transcription when the cell expresses T7 RNA polymerase. Be sure you are using a cell strain that is compatible with T7 expression vectors. <br>
+This is a mutant T7 promoter that includes a lacO operator site. The promoter is inhibited by the LacI protein and can be induced by IPTG. The promoter sits in the 5' region of a gene and initiates transcription when cells express T7 RNA polymerase. Please use cell strains that are compatible with T7 expression vectors. <br>
-The following parameters were calculated and compared against the "wild-type"</html> [https://parts.igem.org/part:BBa_K613007] <html> The following parameters were calculated:
+The following parameters were calculated and compared against the "wild-type" <a href="https://parts.igem.org/part:BBa_K613007">promoter</a>.
-<table border="1">
+<table border="1">
 <tr>
 <th>Promoter</th>
@@ Line 31: / Line 31: @@
 </table>
 <br>
-The cells in this experiment were grown in M9 media without casamino acids. This keeps the cells alive but inhibits significant growth. The fluorescence intensities were still normalized to optical density.
+Cells were grown in M9 media without casamino acids. Fluorescence intensities were normalized to optical density (absorbance @600nm).
 <br>
 <p><img src="https://static.igem.org/mediawiki/igem.org/d/d0/CMU_FAP-MG2.jpg", width="689", height="384"><br>
-Fluorogen activating proteins were used as protein reporters in our characterization experiment as described <a href="http://2012.igem.org/Team:Carnegie_Mellon/Met-Overview">here</a>. Here, a saturating amount of malachite green was added to each well of a 96 well plate and fluorescence values were recorded over time. Our <a href="http://2012.igem.org/Team:Carnegie_Mellon/Mod-Overview">model</a> was used to describe the behavior of our time lapse data using differential equations and fitting algorithms.
+Fluorogen-activating proteins were used as protein reporters in our characterization experiment (see details in <a href="http://2012.igem.org/Team:Carnegie_Mellon/Met-Overview">here</a>). 10uM malachite green was added to each well of a 96-well plate and fluorescence intensities were recorded over time. Our <a href="http://2012.igem.org/Team:Carnegie_Mellon/Mod-Overview">model</a> was used to simulate the time lapse data using differential equations.
 <br><br>
 <img src="https://static.igem.org/mediawiki/igem.org/5/5c/CMU_Spin-DFHBI2.jpg", width="689", height="384"><br>
-The Spinach aptamer was used as an RNA reporter in our characterization experiment as described <a href="http://2012.igem.org/Team:Carnegie_Mellon/Met-Overview">here</a>. Here, a saturating amount of DFHBI was added to each well of a 96 well plate and fluorescence values were recorded over time. Our <a href="http://2012.igem.org/Team:Carnegie_Mellon/Mod-Overview">model</a> was used to describe the behavior of our time lapse data using differential equations and fitting algorithms.<br>
+The Spinach aptamer was used as a RNA reporter in our characterization experiment (see details in <a href="http://2012.igem.org/Team:Carnegie_Mellon/Met-Overview">here</a>). 200uM DFHBI was added to each well of a 96- well plate and fluorescence intensities were recorded over time. Our <a href="http://2012.igem.org/Team:Carnegie_Mellon/Mod-Overview">model</a> was used to simulate the time lapse data using differential equations.
-<br>
+<br><br>
-Note: Not included here are the properties of our characterization method. To learn more about how we characterized this set of promoters, <a href="http://2012.igem.org/Team:Carnegie_Mellon/Met-Overview">click here</a>.
+Note: To learn more about how we characterized this set of promoters, <a href="http://2012.igem.org/Team:Carnegie_Mellon/Met-Overview">click here</a>.
 </p>
 <p>Discussion:<br>
-Mutant III of this set of T7/lac promoters produces a lot of protein and seems to respond efficiently to the addition of IPTG. The design of this mutant was based on a different class of T7 promoters, which are weaker than the traditional variants that are popular in molecular biology. As a result, this promoter is expected to perform weaker than the wild type. We hypothesize that this promoter does not burden the cells as much as traditional T7 promoters that can overload cells and cause selective pressure, among other things.
+The design of this mutant T7Lac promoter (BBa_K921002) was based on a different class of T7 promoters, which are weaker than the wildtype T7Lac promoter (BBa_K613007). Therefore, this promoter is expected to produce less protein than the wildtype promoter. However, this mutant promoter produces more protein than the wildtype promoter in our experiments. We hypothesize that this promoter does not burden cells as much as the wildtype T7 promoters, hence giving rise to higher translation rate of mRNA.
-<br><br> It is important to note that across all three promoters, multiple dips were recorded in the readings even though our data is normalized against optical density to take into account viable cell number. This is due to the fact that strong promoters cause selective pressure and multiphasic growth curves. This can cause sinusoidal behavior in gene expression. The approximate frequency of protein production across all three promoters is 1 hour<sup>-1</sup>.
+<br><br>
+We note that multiple dips were recorded in the readings of all three mutated promoters. This could be due to a strong metabolic burden and multiphasic growth of bacteria.
 </p>