Team:Heidelberg/Contribution


Contribution





Characterized parts

Contribution β-agarase Part:BBa_K2094002

As described in our selective advantage section, we aim to substitute the common antibiotic selection procedure by replacing it with a selective advantage through dietary supplements to enable biomedical compatibility.

For this purpose we provided E. coli with the β-agarase gene that gives the transformed strain the ability to degrade agar. We made use of the β-agarase sequence uploaded for the part BBa_K2094002. Unfortunately there was not much information on the registry page so we decided to further characterize it.

We developed an assay to measure the enzyme activity and published our results on the registry page in order to give further iGEM teams an easy toolset for ß-agarase characterization and measurements in vivo and in vitro.

Briefly, agarase activity was determined using the 3,5-dinitrosalicylic acid (DNS) method [1]. The experimental setup was as follows:

Figure 1 Experimental setup overnight cultures. Overnight cultures of E. coli BL21 with pET15b-β-agarase and E. coli BL21 with pET15b-mCherry were grown at 37 ° in LB medium. To the overnight cultures as well as to the in vivo experiments, carbenicillin was added for selection and IPTG was added to induce expression. Samples were incubated for 12 h at 37 °C with 70 rpm shaking. E. coli BL21 with pET15b-mCherry were used as a negative control to confirm that the occurrence of reducing sugars is due to the β-agarases and not to other metabolic pathways. By using bacteria having the same plasmid but with another insert, a possible influence of the pET15b vector can also be ruled out. Created with BioRender.com

A standard curve of D-galactose dissolved in LB medium was used to determine the total amount of reducing sugars.
Figure 2: results β-agarase activity. (A) Samples from left to right: agarase no agar, agarase in vitro, agarase in vivo,neg. control no agar, neg. control in vitro, neg. control in vivo (B) Quantitative measurement results regarding the standard curve

The expression vector we used possesses a lac operator, therefore we wanted to test the enzyme activity in dependence from expression induction with IPTG as well. The results in figure 3 B show that the values for in vitro measurements are unexpected because measured absorbance and calculated concentrations differed strongly while having the same experimental conditions. The calculated concentration for +IPTG in vitro (0.628 mg/mL) (experiment 2) is high compared to the previous experiment in vitro (0.377 mg/mL) (experiment 1). Nevertheless, a notable influence of IPTG on β-agarase expression can be observed by comparing the results from the in vivo measurements. The β-agarase activity, determined by the amount of reducing sugars produced, is the highest when IPTG is added to the overnight culture as well as to the dilution (in vivo) with a concentration of 0.658 mg/mL. The second highest concentration (0.330 mg/mL) can be observed in the overnight culture where IPTG was added, but lacking in the dilution (−IPTG in vivo). The lowest concentration (0.213 mg/mL) can be observed (0.213 mg/mL) when there is no IPTG neither in the overnight culture nor in the dilution (−IPTG in vivo).

Figure 3: Results influence of IPTG activation on the enzyme activity. (A) Samples from left to right: +IPTG supernatant , +IPTG in vitro, +IPTG in vivo, −IPTG supernatant, −IPTG in vitro, −IPTG in vivo. (B) Measurements: values for “in vitro” from experiment 1 and “in vitro + IPTG” differ noticeably even if experimental conditions were the same.

This shows that the repression of expression in the lac operator does not function with one hundred percent efficiency. The values for reducing sugars without addition of IPTG are 0.213 mg/mL, which is significantly higher than those of the negative control (0.0412 mg/mL). Enzyme activity can be measured even without an inducer, i.e. the β-agarase gene is expressed even without the addition of IPTG, but to a significantly smaller extent. This suggests leakage in the plasmid system.

Contribution AvPAL Part: BBa_K1983000

Another central aspect of our project is the treatment of Phenylketonuria by using phenylalanine ammonia lyase (PAL) as a therapeutic drug. We therefore used the sequence and the information from the part BBa_K1983000. We showed reproducibility of the results from the previous iGEM team regarding its appearance in the SDS page and Western-Blot. We further developed an additional characterization assay based on in vitro enzyme activity measurements.
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.
We controlled the appearance of AvPAL in an SDS-PAGE as well as in a western blot (see Fig. 4). 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.

Figure 4 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. This staining is performed to control if the blotting was successful. The protein bands are visually detectable. (C) shows the final results after the western blot staining with anti-his tag antibodies. The stained protein size was at about 65 kDa, which corresponds to the expected size of our target protein AvPAL.

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. 5). The supernatant was taken to measure the degradation of phenylalanine (Phe) to trans-cinnamic acid (tCa). We used three different phenylalanine concentrations, where 1 mM and 0,5 mM Phe were best detectable (see Fig. 5 D). 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 (see Fig. 5 C).

Figure 5. Absorbance of Phe and tCa and measurments in vitro (A-C) represent the absorbance spectrum of (A) phenylalanine (Phe), (B) trans-cinnamic acid (tCa) and in (C) the merged graphs of (A) and (B). (D) shows the in vitro measurements with the concentration of 1 mM and 0,5 mM Phe added as well as the blank supernatant without any Phe (LB) and the negative control supernatant.

As can be seen in figure 5 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 linear 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 larger 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.
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.

More detailed explanations and experimental results can be also found on our wiki page AvPal Experiments and on the corresponding registry page.

References

[1]G. L. Miller. Use of Dinitrosalicylic Acid Reagent for Determination of Reducing Sugar. Analytical Chemistry. Vol. 31(3):426-428. DOI: 10.1021/ac60147a030