From 2014.igem.org

Revision as of 20:47, 16 October 2014

Results / Heavy metals / Quantitative analysis of our T4MBP

Labwork

The labwork was executed by a Jan Thieleke a staff member of the institute of inorganic chemistry using a ICP-OES. More background about this analysis.

Results

In our project we wanted to construct a protein which binds different heavy metals simultaneously. Therefor we designed a fusionprotein with different binding domains (T4MBP). These domains were expected to bind cadmium, arsenic, zinc and copper. To improve the formation of disulfide bonds we decided to use Origami2 E. coli strain for protein expression. To analyse the binding capacity and function of our protein we decided to use optical emission spectroscopy (OES) and mass spectrometry (MS).

First of all we did growth assays to analyse non-lethal heavy metal concentrations for E. coli. Because of the high toxicity of arsenic, we decided to exclude it from our measurements. For the analyses we grew large scale E. coli cultures with non-lethal heavy metal concentration in the media. At high optical density the cultures were pelleted. The pellets were dried to calculate the dryweight. The dried pellets were solubilized in HNO3 and under high pressure and the samples were analysed via OES. To quantify the effect we compared the results of cells expressing T4MBP to cells without the protein.
Figure 1 shows the detected weights of heavy metals per kg dried bacteria pellets.

Fig 1: measured heavy metals in E. coli pellets E. coli was grown in heavy metal containing media. Cell pellets were dried and analysed via optical emission spectroscopy. WT: wild type Origami2 strain. T4MBP: Origami2 strain expressing T4MBP. Cd: Cadmium, Zn: Zinc, Cu: Copper. Shown are mean values of two replicates. Confidence levels are marked.

Figure 1 points out that the efficiency of binding heavy metals differs for the experiments. For each heavy metal four analyzes were done, they have the same order of bar heights: The lowest bar is always the one with the wildtype and without added heavy metal, meaning the least heavy metal was bound in these experiments. Second lowest bars are the ones with the expressed T4MBP and without added heavy metals. The bars for the wildtype plus one heavy metal are higher, therefore the bacteria itself binds heavy metals, probably up to a lethal dosis. The highest bars are the ones with expressed T4MBP plus heavy metal. Accordingly bacteria bind even more heavy metal, if the T4MBP is expressed. The four bars for copper differ the least and are altogether the smallest, concluding that copper is even in little dosis toxic for bacteria. This conclusion doesn’t apply for zinc and cadmium, due to the much higher and differing bars.
Figures 2 – 4 show the heavy metal binding efficiency difference between wildtype bacteria without added heavy metal and bacteria plus T4MBP and added heavy metal.

Fig. 2: measured heavy metals in dry bacteria pellets of wildtype bacteria (WT) without zinc (Zn) and for bacteria with T4MBP plus zinc.	Fig. 3: measured heavy metals in dry bacteria pellets of wildtype bacteria (WT) without cadmium (Cd) and for bacteria with T4MBP plus cadmium.	Fig. 4: measured heavy metals in dry bacteria pellets of wildtype bacteria (WT) without copper (Cu) and for bacteria with T4MBP plus copper.

Figure 2 shows that through expressed T4MBP about four times more zinc can be bound to bacteria than to the normal wildtype. And at last figure 4 shows the efficiency of binding about 2 times more zinc by T4MBP. A difference of binding cadmium of about 3 times more by T4MBP than without can be seen in figure 3. Additional analyses via mass spectrometry showed 3 times enhanced cadmium binding as well (fig. 5).

Fig. 5: measured heavy metals in dry bacteria pellets of wildtype bacteria (WT) without cadmium (Cd) and for bacteria with T4MBP plus cadmium. Measurement was done with mass spectrometry.

We therefore conclude:

1. The expression and right folding of T4MBP works.
2. Bacteria with expressed T4MBP bind effectively more heavy metals out of the surrounding than wildtype bacteria.
3. The binding of heavy metals works best for zinc, second cadmium and third copper (among of its lethal effect).
4. For arsenic there is no statement possible, but in consideration of points 1-3 we assume that expressed T4MBP will bind arsenic too.