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From 2014.igem.org

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.
We prepared our samples as described in this protocol.

Results

In our project we wanted to construct a protein which binds different heavy metals simultaneously. Therefore 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 Origami 2 E. coli strain for protein expression. To analyze the binding capacity and function of our protein, we decided to use ICP-OES.
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 concentrations 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 under high pressure and temperatures. Afterwards, the samples were analysed via ICP-OES. To quantify the effect of our T4MBP, we compared the results of cells expressing T4MBP to cells without this protein.
Figure 1 shows the detected weights of heavy metals in mg 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 analyzed via optical emission spectroscopy. WT: wild type Origami 2 strain. T4MBP: Origami 2 strain expressing T4MBP. Cd: Cadmium, Zn: Zinc, Cu: Copper. The mean values of two replicates are shown here. Confidence levels are marked.

Figure 1 points out that the efficiency of binding heavy metals differs for the experiments. Because of the small amount of dry pellet, only half-quantitative determinations for the samples of the bacteria consisting T4MBP and added heavy metal were possible. For each heavy metal four analyses were done, which 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, probably because of the small sample amount and of the copper-toxicity for the bacteria. Therefore, no exact statement for the copper binding effiency is possible. This toxicity conclusion doesn’t apply for zinc and cadmium due to the much higher and differing bars.
Figures 2 and 3 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 cadmium (Cd) and for bacteria with T4MBP plus cadmium. Measurement was done with mass spectrometry.

Figure 2 shows that through expressed T4MBP about four times more zinc can be bound to bacteria than to the normal wildtype. 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. 4).

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.