Team:TU Delft/Project/solubility/characterization

From 2010.igem.org

(Difference between revisions)
(Our Emulsifier Assay)
 
(26 intermediate revisions not shown)
Line 1: Line 1:
__NOTOC__
__NOTOC__
{{Team:TU_Delft/frame_check}}
{{Team:TU_Delft/frame_check}}
-
==Characterization of the Hydrocarbon Solubility Parts==
+
<html><center><img src="http://2010.igem.org/wiki/images/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="http://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html>
 +
==Characterization of the Solubility Parts==
-
===Emulsifier Production===
+
===Protein Production Analysis===
-
The ''alna'' gene was induced when the culture reached a density of about 10<sup>8</sup> bacteria per mL. Bacterial and medium samples were taken for sodium dodecyl sulfate (SDS)-gel electrophoresis to monitor AlnA production. The results shown in Fig. ? indicate that expression of AlnA begins ? min after induction and peaks after ? h.
+
The ''alna'' gene was induced when the culture reached a density of about 10<sup>8</sup> bacteria per mL. Bacterial and medium samples were taken for sodium dodecyl sulfate (SDS)-gel electrophoresis to monitor AlnA production. Because the IPTG inducable protomor is generally regarded as verey strong, we expect that the resulting gel wil show when the expression of AlnA begins and when it reaches its maximum.
-
===Emulsifier Assay===
+
===Standard Emulsifier Assays===
-
[[Image:TU_Delft_Rajakumari.jpg|thumb|350px|right|Calibration graph showing a linear increase in absorbance at 660 nm for increasing concentrations of SDS (●). Under the standard detergent assay conditions, various components like 200 mM NaCl (□), 2.0 mM CaCl2 (♦), 10% glycerol (■), 100 μg microsomal membranes (open diamond), 0.2 mM Triton X-100 (▲) and 2.5 mM CHAPS (○) were added and the turbidity was measured. Error bars represent the deviation from five independent experiments and each one performed in duplicate. ''Rajakumari et al'' (2006) Biochemical and Biophysical Methods]]We developed a new assay to measure the emulsification caused by AlnA. Assays currently described in literature involve spectrophotometric measurements of turbidity after mixing. Although many articles show nice graphs (see textbox on the right), we were unable to reproduce them. In our opinion measuring turbidity turned out to be rather arbitrary.
+
[[Image:TU_Delft_Rajakumari.jpg|thumb|350px|right|''Figure 1'']]Besides proving the production of the protein, we wanted to measure the increase of emulsification as well. The only problem was that emulsification is not easily determined. In literature we found many methods that involve spectrophotometric measurements of turbidity after mixing. Although many articles show nice graphs, we were unable to reproduce them. So, in our opinion measuring turbidity is rather arbitrary and there is a clear need for a new emulsification assay.
-
[[Image:TU_Delft_sudan_ii_balls_stick.png|thumb|right|350px|Ball-and-stick model of the Sudan II molecule, an orange-red azo dye used for staining of non-polar substances. Source [http://commons.wikimedia.org/wiki/File:Sudan-II-3D-balls.png Wikipedia]]]
+
'''Example from literature'''
-
That is why, instead of a colorless hydrocarbon, we choose to use the orange Sudan II dye. This molecule stains non-polar molecules and hardly dissolves water. Meanwhile it has a nice absorbance peak at 493 nm, which makes it convenient to measure.
+
Figure 1 on the right shows a calibration graph with a linear increase in absorbance at 660 nm for increasing concentrations of SDS (●). Under the standard detergent assay conditions, various components like 200 mM NaCl (□), 2.0 mM CaCl2 (♦), 10% glycerol (■), 100 μg microsomal membranes (open diamond), 0.2 mM Triton X-100 (▲) and 2.5 mM CHAPS (○) were added and the turbidity was measured. Error bars represent the deviation from five independent experiments and each one performed in duplicate. ''Rajakumari et al'' (2006) Biochemical and Biophysical Methods
-
===Calibration and Background===
+
<html><br style="clear:both;" /></html>
-
[[Image:TU_Delft_emulsification_assay_calibration_curve.jpg|thumb|350px|right|Emulsification Assay calibration curve]]The assay was calibrated using SDS, because this detergent is widely used and makes the outcome of the assay comparable to existing assays. Our measurements are shown in the graph on right. It shows that the assay is very sensible at low SDS concentrations.
+
 +
===Our Emulsifier Assay===
 +
[[Image:TU_Delft_sudan_ii_balls_stick.png|thumb|250px|right|Ball-and-stick model of the Sudan II molecule. Source [http://commons.wikimedia.org/wiki/File:Sudan-II-3D-balls.png Wikipedia]]]
 +
 +
For determining the emulsification capacity of a substance we developed our own emulsifier assay. The main difference to the existing  assays is that instead of emulsifying a colorless hydrocarbon, we added the orange Sudan II dye. This molecule stains non-polar molecules and hardly dissolves in water. The addition of the dye makes measuring the emulsification much easier, because of it’s nice absorbance peak at 493 nm.
 +
 +
Read the [http://2010.igem.org/Team:TU_Delft#page=Notebook/protocols&anchor=Emulsifier_assay Emulsification Assay protocol].
 +
 +
====Calibration====
 +
The assay was calibrated using SDS, because this detergent is widely used and makes the outcome of the assay comparable to existing assays. Our measurements are shown in the graph on right. It shows that the assay is very sensible at low SDS concentrations.
 +
<html><br style="clear:both;" /></html>
 +
[[Image:TU_Delft_Emuls_assay_SDS_calibration.jpg|center|Emulsification Assay calibration curve]]
 +
 +
====Background====
Next, the influence of the culture media was determined. We used LB medium for the initial growth of the cells, and induced the production of AlnA in M9 medium. The background influence of the media on the assay were measured, as shown below. It shows that LB medium already has some emulsification effect, but M9 shows very little influence.
Next, the influence of the culture media was determined. We used LB medium for the initial growth of the cells, and induced the production of AlnA in M9 medium. The background influence of the media on the assay were measured, as shown below. It shows that LB medium already has some emulsification effect, but M9 shows very little influence.
 +
 +
[[Image:TU_Delft_Emuls_assay_M9_LB_background.jpg|center|Background measurements of LB and M9 medium]]
 +
 +
<html><center><img src="http://2010.igem.org/wiki/images/0/00/TU_Delft_project_navigation.jpg" usemap="#projectnavigation" border="0" /></center><map id="projectnavigation" name="projectnavigation"><area shape="rect" alt="Characterization" title="" coords="309,3,591,45" href="http://2010.igem.org/Team:TU_Delft#page=Project/solubility/characterization" target="" /><area shape="rect" alt="Results" title="" coords="609,3,891,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/solubility/results" target="" /><area shape="rect" alt="Parts" title="" coords="9,3,290,44" href="http://2010.igem.org/Team:TU_Delft#page=Project/solubility/parts" target="" /></map></html>

Latest revision as of 13:39, 27 October 2010

CharacterizationResultsParts

Characterization of the Solubility Parts

Protein Production Analysis

The alna gene was induced when the culture reached a density of about 108 bacteria per mL. Bacterial and medium samples were taken for sodium dodecyl sulfate (SDS)-gel electrophoresis to monitor AlnA production. Because the IPTG inducable protomor is generally regarded as verey strong, we expect that the resulting gel wil show when the expression of AlnA begins and when it reaches its maximum.

Standard Emulsifier Assays

Figure 1
Besides proving the production of the protein, we wanted to measure the increase of emulsification as well. The only problem was that emulsification is not easily determined. In literature we found many methods that involve spectrophotometric measurements of turbidity after mixing. Although many articles show nice graphs, we were unable to reproduce them. So, in our opinion measuring turbidity is rather arbitrary and there is a clear need for a new emulsification assay.

Example from literature

Figure 1 on the right shows a calibration graph with a linear increase in absorbance at 660 nm for increasing concentrations of SDS (●). Under the standard detergent assay conditions, various components like 200 mM NaCl (□), 2.0 mM CaCl2 (♦), 10% glycerol (■), 100 μg microsomal membranes (open diamond), 0.2 mM Triton X-100 (▲) and 2.5 mM CHAPS (○) were added and the turbidity was measured. Error bars represent the deviation from five independent experiments and each one performed in duplicate. Rajakumari et al (2006) Biochemical and Biophysical Methods


Our Emulsifier Assay

Ball-and-stick model of the Sudan II molecule. Source Wikipedia

For determining the emulsification capacity of a substance we developed our own emulsifier assay. The main difference to the existing assays is that instead of emulsifying a colorless hydrocarbon, we added the orange Sudan II dye. This molecule stains non-polar molecules and hardly dissolves in water. The addition of the dye makes measuring the emulsification much easier, because of it’s nice absorbance peak at 493 nm.

Read the Emulsification Assay protocol.

Calibration

The assay was calibrated using SDS, because this detergent is widely used and makes the outcome of the assay comparable to existing assays. Our measurements are shown in the graph on right. It shows that the assay is very sensible at low SDS concentrations.

Emulsification Assay calibration curve

Background

Next, the influence of the culture media was determined. We used LB medium for the initial growth of the cells, and induced the production of AlnA in M9 medium. The background influence of the media on the assay were measured, as shown below. It shows that LB medium already has some emulsification effect, but M9 shows very little influence.

Background measurements of LB and M9 medium

CharacterizationResultsParts