Part:BBa_K1602005

Xylose to xylitol converting construct

Xylose is a monosaccharide belonging to the aldopentose family. Through reduction it can be converted to xylitol. The reaction takes place in the cytosol of the host and recent studies show, that the formation of xylitol in E.coli seems possible as well.
To enable the reduction in E.coli it is mandatory to establish an operon containing the coding gene GRE3 for a aldose reductase. The gene is taken from Saccharomyces cerevisiae. The aldose reductase converts xylose to xylitol in dependence of NADPH.

Figure 1 Reaction scheme of the xylose to xylitol converting operon. Xylose is the only substrate needed for the reaction. Xylose is metabolized to xylitol in 1 step in dependance of NADPH.

Usage

This part is a composite of one coding gene and a strong RBS (BBa_B0034) in front of it, under control of a T7 Promoter (BBa_I719005).

aldose reductase - GRE3 (BBa_K1602004)
	Figure 2 Genetic map of the xylose to xylitol converting operon with T7 promoter. This brick enables E.Coli BL21 cells to convert xylose to xylitol in presence of the inductor IPTG.

Results

The expression of GRE3 has been visualized via SDS-PAGE. Positive clones were grown at 37° celsius until an OD of 0,5. Afterwards the cells were induced utilizing 20µl of 1M IPTG for 10h at 28° celsius. Finally the cells were lysated with heat and the suspension was put on the PAGE.

Figure 3 Scan of the PAGE containing from left to right a marker (M; Protein Marker III AppliChem), the positive sample (1) and a negative control (2). The picture was cropped and edited for clarification purposes.	Figure 4 Plot of the gel lanes based on contrast analyses - created with ImageJ

GRE3 assay

To prove the enzymatic activity of the aldose reductase GRE3 in dependance of NADPH we designed an applicable assay as following. We used a spectral analysis with a wavelength of 340nm to make the conversion from NADPH to NADP⁺ observable. A drop in the curve of the absorption spectrum therefore shows that NADPH is being converted to NADP+ i.e. the enzyme works. Spectral analysis was performed with a TECAN® Infinite 200 PRO microplate reader. The resulting data sheets are then put into a plotting script written in R and exported as a ggplot.

The assay was performed as described below:
First Na₂HPO₄ was adjusted to a pH of 7.0 to function as a buffer. The final concentration of Na₂HPO₄ was 0,1M. The assay system contained 0,1mM NADPH and 8 µl were added per well. As a possible blank wells with just NADPH (8 µl to 192 µl of buffer solution) were provided. In addition we added blanks containing just xylitol (0,1M) as well as one containing just NADP+ (0,1mM). The negative control contained a purified TES protein fraction from disrupted BL21 cells. The assay mixture included 154 µl buffer solution, 8 µl xylose, 8 µl NAPDH, and 30 µl of different protein amounts each, ranging from 5µl to 30 µl (in six steps; protein concentration unknown because purification was not performed, just a lysis of the cells with TES). All samples were prepared on ice. (In hindsight the possible blank with just NADPH appears to be a non-optimal solution because the auto catalyzation of this chemical likely happens just a few minutes into the assay.)

The 96 well microplate was loaded as depicted in the picture below:

	The assay was run for 200 kinetic cycles, each 30 secs long and with 25 photo pulses per cycle. The reader was heated to the appropriate temperature of 37° celsius.
Figure 5 96-well microplate layout
Figure 6 The plot on the right hand side is showing the change in absorption of the bromothymol blue in solution for the three different kinds of samples in correlation to the kinetic cycles i.e time. The 'p' curve shows the absolute conversion of educt to product (product curve). The 'ca' curve shows the enzymatic activity of cadA. The activity explains the steady rise of the curve which comes from the acidification of the assay mixture in those wells. Because of the conversion from cis-aconitate to itaconic acid and the byproduct carbon dioxide (which forms bicarbonate acid) and lowers the pH The 'k' curve shows the negative control containing TES protein fraction without cadA. The sudden drop of the 'p' curve in the beginning of the measurement could be explained through various reasons. One could be that the bubbles that formed in the wells when adding the chemicals could interfere with the light. Also the different substances were added on ice so when the plate was put in the heated reader water in the air might have condensated at the bottom of the wells and hinder the light beam. The resulting vibrations of shutting doors too harshly might also interfere with the plate moving mechanism inside the reader and cause malfunctioning.

The code utilized to render the plots is embedded below.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Unknown
• 21
  INCOMPATIBLE WITH RFC[21]

  Unknown
• 23
  INCOMPATIBLE WITH RFC[23]

  Unknown
• 25
  INCOMPATIBLE WITH RFC[25]

  Unknown
• 1000
  COMPATIBLE WITH RFC[1000]