Team:Edinbrugh/Results/PcstA-xylE

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Latest revision as of 02:01, 30 October 2008

PcstA Reporter Assay

Background and Aims

We have created a BioBrick^TM of the cstA promoter (P_cstA) as a possible promoter for our cellulase construct. According to the literature, P_cstA is suppressed by high concentrations of glucose. We expect this property to be useful in our system as cellulolysis will be activated when the glucose supply is depleted.

This assay was designed to determine:

Whether our P_cstA BioBrick^TM is functional.
Whether our P_cstA BioBrick^TM is suppressed by high concentrations of glucose and other monosaccharide and disaccharide sugars.

Procedure

xylE as reporter gene was added to the PcstA BioBrick^TM. JM109 E. coli cells were transformed with this construct.
Cells were grown overnight in LB containing:
1. No added sugar,
2. 5, 10, 20 or 40mM of glucose,
3. or 40mM of either arabinose, lactose or maltose.
Cells were resuspended in 50mM Tris buffer, pH 7.5, prior to assay to control for pH differences between the various cultures.
Cells were developed with 0.5mM catechol for 5 minutes.
The xylE gene product converts colourless catechol into a yellow oxidation product, which can be quantified by measuring absorbance at 375nm ([http://jb.asm.org/cgi/content/abstract/171/12/6617 Ingram et al.]). We measured absorbance at 377nm (A377).

Results

These results indicate that:

Our P_cstA BioBrick^TM is functional. High quantities of the xylE gene product were generated in the absence of sugars.
The promoter activity of P_cstA is inversely proportional to the concentration of glucose. P_cstA is extremely sensitive to glucose: there is a dramatic drop in promoter activity between glucose concentrations of 0mM and 10mM.
P_cstA is sensitive to glucose, arabinose and maltose. However, glucose suppresses P_cstA most strongly and at the lowest concentrations.
Note that the failure of lactose to cause repression is probably due to the fact that our host strain, E.coli JM109, is not able to degrade lactose due to the lacZ-delta-M15 mutation required for blue-white selection.

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-'''[[Team:Edinburgh/Results/Glycogen3|< Previous Entry]] | [[Team:Edinburgh/Results|Tests and Results Index]] | [[Team:Edinburgh/Results/PcstA-xylE|Next Entry >]]'''
+'''[[Team:Edinburgh/Results|Back to Results]]'''
-== PcstA Reporter Assay ==
+== P''cstA'' Reporter Assay ==
 === Background and Aims ===
-We have created a BioBrick of the ''cstA'' promoter (PcstA) as a possible promoter for our cellulase construct. According to the literature, PcstA is suppressed by high concentrations of glucose. We expect this property to be useful in our system as cellulolysis will be activated when the glucose supply is depleted.
+We have created a BioBrick<sup>TM</sup> of the ''cstA'' promoter (P<sub>''cstA''</sub>) as a possible promoter for our cellulase construct. According to the literature, P<sub>''cstA''</sub> is suppressed by high concentrations of glucose. We expect this property to be useful in our system as cellulolysis will be activated when the glucose supply is depleted.
 This assay was designed to determine:
-* Whether our PcstA BioBrick is functional.
+* Whether our P<sub>''cstA''</sub> BioBrick<sup>TM</sup> is functional.
-* Whether our PcstA BioBrick is suppressed by high concentrations of glucose and other monosaccharide and disaccharide sugars.
+* Whether our P<sub>''cstA''</sub> BioBrick<sup>TM</sup> is suppressed by high concentrations of glucose and other monosaccharide and disaccharide sugars.
 === Procedure ===
-# ''xylE'' as reporter gene was added to the PcstA BioBrick. JM109 ''E. coli'' cells were transformed with this construct.
+# ''xylE'' as reporter gene was added to the P''cstA'' BioBrick<sup>TM</sup>. JM109 ''E. coli'' cells were transformed with this construct.
 # Cells were grown overnight in LB containing:
 ## No added sugar,
@@ Line 29: / Line 29: @@
 These results indicate that:
-* Our PcstA BioBrick is functional. High quantities of the ''xylE'' gene product were generated in the absence of sugars.
+* Our P<sub>''cstA''</sub> BioBrick<sup>TM</sup> is functional. High quantities of the ''xylE'' gene product were generated in the absence of sugars.
-* The promoter activity of PcstA is inversely proportional to the concentration of glucose. PcstA is extremely sensitive to glucose: there is a dramatic drop in promoter activity between glucose concentrations of 0mM and 10mM.
+* The promoter activity of P<sub>''cstA''</sub> is inversely proportional to the concentration of glucose. P<sub>''cstA''</sub> is extremely sensitive to glucose: there is a dramatic drop in promoter activity between glucose concentrations of 0mM and 10mM.
-* PcstA is sensitive to all sugars that were tested. However, the degree to which PcstA is sensitive to each sugar differs: glucose > maltose > arabinose > lactose.
+* P<sub>''cstA''</sub> is sensitive to glucose, arabinose and maltose. However, glucose suppresses P<sub>''cstA''</sub> most strongly and at the lowest concentrations.
+* Note that the failure of lactose to cause repression is probably due to the fact that our host strain, ''E.coli'' JM109, is not able to degrade lactose due to the ''lacZ-delta-M15'' mutation required for blue-white selection.