Team:Freiburg/Results/Cellfree

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Results: Cell-free Expression

The purpose of cell-free expression in the DiaCHIP is to copy a DNA template into a protein microarray on demand. To enable antibody detection with this protein microarray, the single antigen spots have to be covered with a dense layer of antigens. Thus, the expression efficiency of the cell-free system has to be optimized to produce a sufficient amount of the target proteins within a timespan that is reasonable for DiaCHIP preparation in the suggested applications (LINK).

One of the key factors influencing expression efficiency is the design of the expression vector. Therefore, we started with the design of a vector based on pSB1C3 that would allow successful cell-free expression (LINK). Additionally, we received an expression vector containing a GFP coding sequence form the BIOSS toolbox (LINK/MAP). The third vector we used for our experiments was pBESTluc (LINK/MAP) encoding a luciferase for performing the Luciferase assay.

However, the most important thing is of course the expression system itself. We obtained one commercially available expression kit based on an //E. coli// lysate, were provided with an established cell-free expression mix from the group of Bernhard (LINK!!) and additionally established a protocol for the production of such a system ourselves, based on [REF.]. We named it the DiaMIX and provide the protocol in order to give future iGEM Teams the possibility to produce their cell-free expression mix in a low-budget version themselves.

First successful expression

Figure 1: Cell-free expression of GFP. Fluorescing GFP could be detected via fluorescence microscopy after cell-free expression (A). Picture taken of the negative control (B). For detailed reaction performance see our labjournal.

Before using our DiaMIX in the microfluidic chamber, we performed some initial experiments //in vitro// in well-plates. With our second cell-free expression experiment using all our self-prepared components we were already able to express GFP deriving from pQE60 (LINK). As negative control the reaction was performed in the same way, simply using water instead of DNA. After expression, a small amount of the reaction was directly pipetted onto a microscopy glass slide and analyzed under a fluorescence microscope. As it is clearly visible in figure 1, we could detect expressed GFP and therefore prove the functionality of our system for the first time!

Variation of Mg(OAc)2 concentration

Figure 2: Impact of Mg(OAc)2 addition during cell-free expression of tYFP. Mixtures of our own lysate with our premix (KK) and the lysate we obtained from Bernhard with our premix (BK) were tested. An impact of feeding with Mg(OAc)2 could be observed for the BK mixture. Validation of fluorescence at an excitation of 488nm.

Nonetheless, there were of course many things that had to be optimized before performing the cell-free expression on a slide for a measurement with the iRIf (LINK was ist iRIF) device. At first, we investigated the influence of magnesium acetate (Mg(OAc)2) added to the reaction. In a work of ???? (LINK) we read about the enhancement of cell-free expression by regularly adding a particular amount of the chemical. We performed some expression experiments where some reactions were fed with small amounts of Mg(OAc)2 and some were not. Indeed, we could observe an effect of the addition of the supplement (see figure 2). However, the actual impact of the feeding varied a lot in the experiments (LINK zu labjournal), even among reactions with the same mix components.



Figure 3: Variation of Mg(OAc)2 concentrations in cell-free expression. Cell-free expression of pBESTluc and subsequent validation via Luciferase assay.

As supplying a reaction lasting for two hours with some additional chemicals every 20 minutes is quite time consuming, we thought about simply varying the initial concentration of Mg(OAc)2 in the reaction mix. For the expression mix we received from Bernhard, the optimal start concentration was stated in the range of 15-18mM (REF!!). Therefore, we expressed the pBESTluc (LINK) with our whole DiaMIX at concentrations between 14-18mM to compare the expression via Luciferase assay. The result is shown in figure 3 and reveals the optimal amount of Mg(OAc)2 at a starting concentration of 14mM.



Variation of DNA concentration

After establishing the best concentration of Mg(OAc)2 in our reactions we further investigated the optimal concentration of DNA added for expression. Again, pBESTluc (LINK) was expressed to allow the analysis via Luciferase assay in a microplate reader. Triplicates for reactions of 50µl volumes were set up with amounts of DNA ranging from 1µg to 5µg, as well as a negative control without DNA. Exemplarily, the results for three different concentrations as well as the negative control are shown in figure 4, the other results can be found in our labjournal. The optimal amount of DNA established for further experiments was 2µg.

Figure 4: Variation of DNA concentration for cell-free expression. Results for concentrations of 0.02µg/µl (A), 0.04µg/µl (B), 0.1µg/µl (C) and the negative control (D). Validation was performed via Luciferase assay and analyzed in a microplate reader.


Comparison with commercial kit

Figure 5: Cell-free GFP expression over time. Comparison ot the commercial kit with the DiaMIX was performed using the pQE60 HA-GFP-His vector. Relative fluorescence was measured every minute over 2 hours. As negative control the fluorescence of the respective mix without adding DNA was recorded.

Having optimized our self-prepared low-budget cell-free expression mix, we were keen on comparing it to a commercially available kit and thereby making a statement about the functionality of our DiaMIX. As already shown on the main results page we set up an experiment using DNA of the pQE HA-GFP-12xHis vector in order to express functional GFP and analyze it via fluorescence. The cell-free expression itself as well as the negative control for each mix were prepared in triplicates.
Our self-produced DiaMIX performed about as good as the commercial kit as it is shown in figure 5. Compared to the background, an expression time of two hours resulted in a 2-fold increase in relative fluorescence in both systems. The background fluorescence was estimated by the negative control.