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.

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 tubes or at a later timepoint in well-plates. First we weren't able to express anything. This was due to several factors, which will be explained shortly. So for example the correct Ph is of utmost impotance and has to be adjusted in an relativley narrow window between 7 and 8, which isn´t an easy task with only pH-Paper as tool. The pH is also crucial for the solubility of certain chemicals. After adjustment of pH further Parameters were adapted and the source of error narrowed down. As prime sources for errors the Lysate and the premix were identified. Repeated preparataion and gain of experience took care of that.

In the end our Premix was our greatest error source and freshly prepared. This resulted in some low basic expression. This called for some further optimization of our system.

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.

At first, we investigated the influence of magnesium acetate (Mg(OAc)₂) added to the reaction. In a work of Kim et al. (LINK) we read about the enhancement of cell-free expression by regularly adding a particular amount of this chemical. We performed some expression experiments where some reactions were fed with small amounts of Mg(OAc)₂ and some were not. The repeated addition of small amounts of magnesium (100 nmol) every 20 minutes during the reaction was found to increase expression levels and was therefore implemented. This was done simply by pipetting 0.5 μl of a 10 μM magnesium acetate solution into the reaction tube whilst keeping it inside the thermomixer. Positive effects towards expression were immediately visible in a subsequently conducted luciferase assay. 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)>sub>2 in the reaction mix. For the expression mix we received from Bernhard, the optimal start concentration was stated in the range of 15-18 mM (REF!!). Therefore, we expressed the pBESTluc (LINK) with our whole DiaMIX at concentrations between 14-18 mM 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 14 mM.

Variation of DNA concentration

Figure 3: Variation of DNA concentration for cell-free expression. Results for concentrations of 0.02 µg/µl (A), 0.04 µg/µl (B), 0.1 µg/µl (C) and the negative control (D). Validation was performed via luciferase assay and analyzed in a microplate reader.

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. For this, a sequence of reactions was performed in parallel, reaching from 1 to 5 μg (per 50 μl reaction). Each concentration was performed in a triplicate. As a negative control, the same conditions (37°C, 2 h, no shaking) applied to a reaction with water added instead of DNA. Luciferase was expressed using the pBESTluc vector and a luciferase assay was performed as described inprotocols. The optimal amount of DNA established for further experiments was 2 µg or 3 µg. To make cost-efficient reactions for further reactions 2 µg DNA per 50 µl were used.

Figure 4: Variation of DNA concentration for cell-free expression. Results for concentrations of 0.02 µg/µl (A), 0.04 µg/µl (B), 0.1 µg/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. The experiment was performed in triplicates and the values normalized to air. A propagation of uncertainty was performed.

Figure 5: Cell-free GFP expression over time. Comparison ot the commercial kit with the DiaMIX was performed using the self-developed Spy-GFP-10His. Relative fluorescence was measured every minute over 2 hours. As negative control the fluorescence of the respective mix without adding DNA was recorded. The Experiment was performed in triplicates and the values normalized to air. An propagation of uncertainty was performed.

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 our external standard that had already demonstrated to perform well in cell-free expression system. Furthermore a from this years iGEM team especially for the application designed vector was used that carried a gene for GFP and an N-terminal 10X His and a Spy-tag on the other side. Non-coding areas surrounding the GFP gene were optimized for cell free expression. (link) As can be seen in Figure 5 The DiaMIX shows a higher fluorecence compared to the commercial kit. Even when the higher groundlevel fluorescence is taken into account. Which is an effect of the components in the premix. The commercial kit shows an initial drop in fluorescence. This was observed during every measurement and seems to be an fundamental characteristic of the mix that we attributed quenching effects. Figure 6 shows the performance of our self-designed vector with tha DiaMIX and the comercial mix. As can be observed the DiaMIX still performs better than the bought kit. However the difference is not as obvious as before. This indicates that the DNA does not posses the optimal structure jet for an effective performance in a cell-free system. This could be due to differences in the 5' upstream sequence which was demonstrated to have great influence on the initiation of transcription. Furthermore, the tag in the beginning of the coding sequence also manipulates the expression level as shown by ???
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.

iRIf measurement of cell-free expressed GFP

Figure 6: iRIf measurement of cell-free expressed GFP.

Encouraged by all these promising results obtained we decided to make the next step and broaden the verification methods of our cell-free expressed proteins by using the iRIf technology. We validated cell-free expressed GFP by pipetting a small amount of the reaction directly onto a glass slide with different surfaces. We checked for the detectability of the protein as well as for the results on specific and unspecific surfaces.
We were able to detect GFP expressed with the DiaMIX as it can be seen in figure 6. Elaborate results for the iRIf experiments performed with cell-free expressed proteins can be found on this results page.

On-slide cell-free expression

The last step to the final application in the DiaCHIP is the performance of the cell-free expression reaction between the silicone slide and the glass slide of the microfluidic chamber. Immobilized DNA on the PDMS slide should be transcribed and translated into protein. Due to time constraints, we were not able to optimize this process. Nonetheless, you can find some initial results on this results page.