Revision as of 09:00, 19 October 2021

Codon optimized AvPAL with C-terminal 6XHis-Tag

Overview

Phenylalanine ammonia lyase (PAL) from Anabaena variabilis is an enzyme that catalyzes breaking down L-phenylalanine to ammonia and trans-cinnamic acid. This part was introduced by Vilnius-Lithuania iGEM Team as a functional part used to break down L-phenylalanine in vivo. This is an improved Stanford-Brown 2013 AvPAL biobrick part. We have removed the T7 promoter and replaced the N-terminal FLAG-tag with a more common C-terminal 6xHis-tag. The C-terminal tag location was chosen assuming it would less likely affect the stability and folding of the protein.

Our team improved the characterization of this part by testing the enzyme's activity in vivo. We also have experimentally measured the expression rate and time to reach steady of the protein in Escherichia coli cells, since the kinetic constants of this enzyme are determined [1]. Expression of the 6XHis-tagged protein is easily detectable via Western Blot. We used pBAD expression system for our project and characterization of this part.

Experiments and Results

PAL was cloned into pET and pBAD expression vectors, with the latter showing better expression results. Thus, further experiments were carried out using pBAD vector and the procedures below were described using this vector (see results page for more information).

Cloning

The received sequences were amplified using PAL-Chis FW/RV primers and digested with Esp3I and XhoI. The fragments containing mutant genes were cloned into pBAD expression vector digested with NcoI and XhoI. Transformant colonies were PCR-screened using pBAD-Pro/Term primers and positive pBAD PAL clone plasmids were sequenced prior to further usage.

Expression assays

SDS-PAGE and Western Blot

pBAD PAL expression was tested in E. coli TOP10 strain, showing positive results on SDS-PAGE gel and Western Blot (Fig. 1).

Figure 1. A 12% SDS-PAGE analysis and Western blot. PAL C-6XHis tag expression in TOP10 strain induced by L-arabinose (0.2 %, w/v). S and IS denote soluble and insoluble fractions respectively.

PAL expression rate over time

Additionally, PAL expression was tested over time to measure the time needed for PAL to reach a steady state concentration inside the cells (Fig 2 and 3).

Figure 2. PAL expression over time Cells were grown in 6ml LB medium overnight and induced with 0,2% w/v arabinose. Aliquots of 700μl were harvested at certain time points after measuring OD600. The harvested cells were centrifuged at 12,000xg for 10min and the pellet was resuspended in 50μl of 4x SDS-PAGE loading buffer. Samples were heated at 95⁰C for 10min and centrifuged at 12,000xg for 10min. 10μl from the top part of the lysate mixture were loaded into the gel. After electrophoresis, the gel was stained with Coomassie Blue.

PAL activity in vivo results in optimal conditions (see below) can be used in determining the quantities of PAL inside the cells since the kinetic constants of this enzyme are accessible [1]. It was assumed that if surfactants (60% EtOH) eliminated the impermeability [2] of the membrane, the true values of PAL expression inside the bacteria could be calculated from the known kinetic constants. The expression rate over time was normalized by dividing each measured band score by the OD600 at the corresponding time point.

Figure 3. PAL expression over time The graph shows the increasing PAL expression over a period of 4 hours. Approximate quantities were calculated from the PAL activity in vivo results under conditions of EtOH 60% representing the maximum amount of PAL available in the cells. The gel (Fig. 4) was scanned and the appropriate bands representing PAL expression were quantified using ImageJ.

This data is later used in our model of the system, since it describes the time for PAL to reach steady state concentration.

PAL characterizaton in vivo

E.coli expressing PAL was tested under laboratory conditions (Fig. 4) to see if the enzyme is working in vivo. Since PAL is expressed inside the cell, L-phenylalanine has to permeate the membrane and diffuse to the interior. To test the effect of the membrane as a mechanical boundary for L-phenylalanine to pass through, surfactants of varying concentrations were also used. This experiment has proven that E.coli expressing PAL can effectively convert L-phenylalanine to tCA. Also, the effect of surfactants showed that the membrane is one of the limiting factors to the efficiency of the probiotic. The use of surfactants was also helpful to identify the quantities of PAL expressed inside the cells since the membrane boundary effect was eliminated.

Figure 4. Activity of PAL in vivo under different conditions Permeability of the cells was enhanced by exposing them to different concentrations of ethanol and Tween-20. After this, the cells were placed into the reaction mixture for a period of 20 minutes in pH 8.8 (see methods page).

Since the previous experiments showed that in order for PAL to work more effectively, the membrane ruggae had to be increased, so the next step was to test the cooperation of a constantly expressed PheP (E.coli L-phenylalanine permease) from a composite biobrick part (BBa_K1983014) with PAL (Fig. 5). The role of PheP was to facilitate the diffusion of L-phenylalanine to the cell‘s interior, thus increasing the effectiveness of conversion to tCA.

Figure 5. Activity of PAL and PAL with PheP in vivo Activity of PAL and PAL with PheP (BBa_K1983014) in E. coli cell over a period of 20 minutes in 7.4 pH. The activity is evaluated by production of tCA. The initial amount of L-phenylalanine in reaction mixture was 1.1 g. Every system which was tested during this experiment was transformed into E. coli TOP10. Control - E. coli TOP10 strain without PAL and pheP biobricks. The total mass of recombinant cells in the reaction mixture was 5 grams.

This result shows that incorporating membrane transporter for phenylalanine was valuable for the whole systems avtivity in vivo.

Contribution

• Group: iGEM Team Heidelberg 2021
• Author: Franziska Giessler, Silja Malkewitz, Marilena Wittmaack
• Summary: The Part BBa K1983000 was used for our project and further characterized by in vitro enzyme activity measurements.

Experiments and Results

Cloning

The AvPAL DNA with the sequence from the part BBa_K1983000 was cloned into a pET15b backbone using BamHI and NdeI restriction enzymes. We expressed the enzyme in E. coli BL21 with induction through IPTG.

Blotting

We controlled the appearance of AvPAL in an SDS-PAGE as well as in a western blot in order to show reproducible results (see Fig. 6). Additionally, we tried to clone AvPAL into the pUC19 Backbone but it did not work out and therefore can be seen as a negative control in the SDS-PAGE and the western blot.

Expression

The enzyme was expressed in a 50 mL overnight culture. The pellet was lysed in DPBS and bacteria were fracked in a french press machine. After centrifugation, one part of the supernatant and the pellet was used for the SDS-PAGE and the other part of the supernatant for the in vitro measurements (see Fig. 7).

Figure 6. AvPAL SDS-PAGE, Ponceau staining and Western Blot. (A) shows the SDS-PAGE with coomassie blue staining. (B) is the ponceau staining of the PDVF membrane after blotting. (C) shows the final results after the western blot staining with anti-his tag anitbodies.

In vitro assay of enzyme activity

As described above the supernatant of the bacterial fractionation was taken to measure the degradation of phenylalanine (Phe) to trans-cinnamic acid (tCa). We used three different phenylalanine concentrations, where 1mM and 0,5 mM Phe were best detectable (Fig. 7D). Supernatant of the bacteria with AvPAL cloned into the pUC19 Backbone was used as a negative control. The absorbance was measured at 300nm because at this wavelength the absorbance differed clearly between Phe and tCA (Fig. 7C).

As can be seen in Figure 7 (D), the absorbance at 300 nm increases for both, positive control (supernatant of bacterial fractionation with phe added) and negative controls (supernatant of bacterial fractionation without Phe, supernatant of bacterial fractionation AvPAL in PUC19). However, the increase in the negative curves is quite irregular and no saturation is seen. It is therefore likely that this increase is due to other processes or side reactions in the solution. The positive controls show a much greater increase in absorbance at 300 nm with saturation occurring after 4-6 h, as well as it would be expected for the detection of tCa.

Discussion

The measured values indicate that PAL is present as a functional enzyme in our supernatant of the fracked cells, degrading Phe to tCA. After about 4-6 h, no further increase in tCA concentration is seen, indicating complete turnover. As expected, the phase of complete conversion is reached earlier at lower Phe concentration.

References

1. Lovelock, S. L. and N. J. Turner (2014). "Bacterial Anabaena variabilis phenylalanine ammonia lyase: a biocatalyst with broad substrate specificity." Bioorg Med Chem 22(20): 5555-5557. 2. Cui, J. D., S. R. Jia, et al. (2008). "Influence of amino acids, organic solvents and surfactants for phenylalanine ammonia lyase activity in recombinant Escherichia coli." Lett Appl Microbiol 46(6): 631-635.

Sequence and Features