Team:TU Darmstadt/Project/Bio/Monomeres/Results

Results

Abstract

Hereafter we describe and present our achievements considering the characterization of our proteins:

  • SDS-PAGEs
  • Protein Assays
  • HPLC

We chose various methods, suiting the various proteins and there specific properties.

Itaconic Acid

SDS-PAGE

The expression of cadA has been visualized via SDS-PAGE. Positive clones were grown at 37° celsius until an OD of 0,7. Afterwards the cells were induced utilizing 20µl of 1M IPTG for 12h at 28° celsius. Finally the cells were lysated via ultrasonic cell disruption.

Figure 1 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 2 Plot of the gel lanes based on contrast analyses - created with ImageJ


cadA Assay

The decarboxylic activity of cadA was shown via a pH indicator assay. As a byproduct of the catalytic conversion of cis-aconitate to itaconic acid carbon dioxide is released into the assay mixture. Here it forms carbonic acid and thereby lowers the pH value of the mixture. This is visualized through the indicator bromothymol blue (BTB) which changes its configuration state depending on the surrounding pH-Value (pH 6.0 - 7.6). That change of configuration can be shown via a photometric analysis in 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 following:
First Na2HPO4 was adjusted to a pH of 7.0 to function as a buffer. The final concentration of Na2HPO4 was 5mM. The assay system contained 10% (v/v) indicator stock (BTB) and 20 ul were added per well. As a negative control a purified TES protein fraction from disrupted BL21 cells was used. In a range from 0mM to 1mM (0,2mM steps; total of 6 reactions) cis-aconitate (substrate), dissolved in Na2HPO4-buffer solution was added per well to enable the enzymatic conversion. To put the turnover into relation with the maximum turnover we added extra rows that contained equal amounts of substrate and product (itaconic acid). Finally a row containing just itaconic acid was put at the bottom to be able to use the resulting values as a blank for the assay. All samples were prepared on ice. The 96 well microplate was loaded as depicted in the picture below:

Figure 5 96-well microplate layout


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. Absorbance was measured at the absorption maximum of BTB which in this case is 620nm.

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.

HPLC

MS Itaconic acid Itaconic acid control with 0,005g/mL itaconic acid

  • Control measurement with 0,005g/mL
  • Itaconic acid molecule peak at 130u
  • HPLC with subsequent MS
  • Cleavage row visualized in image underneath as well as in circled peak values

  • Induced with IPTG→ simultaneously addition of xylose as carbon source
  • HPLC with subsequent MS
  • Itaconic acid molecule peak not detectable →instable molecule →rapid decay
  • Biggest relative abundance at 41u
  • Washing step was conducted with TRIS-HCL→ molecule peak at 121→ seen at TRIS-database-MS


The code utilized to render the plots is embedded here at the bottom of the page.

Ethylene Glycol


SDS-PAGE


After we verified the correctness of the operon sequence it was transformed into E. coli BL21 from which we isolated proteins after induction for 12 hours at 28°C with 1mM IPTG. Through SDS-PAGE we were able to validate the high overexpression of xylB and xylC in E. coli.

Further more we induced E. coli BL21 with IPTG and D-xylose at a concentration of 4g/l. Induction was performed for 12 hours. Afterwards cells were harvested and lysated. The cell lysate was extracted with dichlormethan and measured with HPLC-mass spectrometrie. Unfortunately the lysate did not show the characteristic ethylene glycol peaks. Future characterisation should involve higher xylose concentration or the overexpression of the other enzymes of the pathway.

https://static.igem.org/mediawiki/parts/8/8e/TU_Darmstadt_EG_xylBC_PAGE.png

NAD+-Assay

As xylB is a NAD+ dependent Enzyme, we tried to demonstrate its oxidoreductase character by measuring the absorption of a reaction mixture in a 96-well-plate at 340nm, a sinking absorption showing the reduction of NAD+ to NADH as NAD+s absorption peaks at 340 nm. The change in absorption was measured using a TECAN® Infinite 200 PRO microplate reader. The resulting plots were created in Excel and are shown below. The enzymes were purified via TES The assay was performed as following: 100mM Tris/HCl buffer was adjusted to a pH of 8.2. Substrates for the reaction were a 5mM solution of NAD Na salt in the aforementioned buffer and a 10mM solution of d-xylose in the same buffer. Controls were made with only buffer (200µl), buffer and xylose (190µl buffer, 10µl d-xylose), buffer and NAD+(195µl buffer, 5µl NAD+ solution), buffer and enzyme (190µl buffer, 10µl NAD+) and buffer, enzyme and xylose(180µl buffer, 10µl d-xylose solution and 10µl enzyme). The assay was run over 300 cycles, with measurements every 30 seconds at a constant temperature of 30°C. https://static.igem.org/mediawiki/parts/9/9a/T7-xylB-xylC_Assay.png

HPLC

Ethylene glycol - Control standard

  • Gas chromatography with subsequent MS
  • Molecule peak at 62.1u
  • Highest relative abundance at 31u→alpha-decay

Selfmade control with addition of pure ethylene glycol to solubilized induced cells

  • Gas chromatography with subsequent MS
  • Molecule peak at 62.1u
  • Highest relative abundance at 31u→alpha-decay
  • Contamination in relation to standardized control visible
  • Induced with IPTG → simultaneously addition of xylose as carbon source

Induced sample ÜS1

  • Gas chromatography with subsequent MS
  • Molecule peak expected at 62.1u→not visible→alpha decay product not visible as well→ethylene glycol not contained within sample
  • Highest relative abundance at 27.9u→Contamination with cell intermediates
  • Contamination in relation to standardized control visible
  • Induced with IPTG → simultaneously addition of xylose as carbon source

Xylitol

SDS-PAGE

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 1 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 2 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:

Figure 5 96-well microplate layout

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 6 The plot on the right hand side is showing the change in absorption of NADPH at 340nm in solution for the two different kinds of samples in correlation to the kinetic cycles i.e time.

  • The 'gre' curve shows the enzymatic activity of GRE3. The curve drops later than the 'k' curve because the active conversion of xylose to xylitol in dependance of NADPH happens at a much quicker rate than the auto catalyzation of NADPH itself.
  • The 'k' curve shows the negative control containing just NADPH without GRE3.

HPLC

MS Xylitol

Xylitol standard sample with 1,2 mg/mL xylitol

  • Induced with IPTG→ simultaneously addition of xylose as carbon source
  • HPLC with subsequent MS
  • Xylitol molecule peak at 152u →Not visible on spectre→instable molecule→rapid decay
  • Highest relative abundance at 61u

Induced sample D, concentrated

  • HPLC with subsequent MS
  • Xylitol molecule peak at 152u →Not visible on spectre→instable molecule→rapid decay
  • Induced with IPTG→ simultaneously addition of xylose as carbon source
  • Washing step was conducted with TRIS-HCL→ molecule peak at 121→ seen at TRIS-database-MS

Measurement notes

relative intensity of TRIS

  • TRIS was used for cell washing steps
  • Molecule peak expected at 121u→instable molecule→rapid decay→highest relative abundance at 90.1→detectable in all induced samples
  • Possible overlap with decay scheme of xylitol

itaconic acid decay scheme

xylitol decay scheme

ethylene glycol decay scheme

The code utilized to render the plots is embedded here at the bottom of the page.