Team:MADRID UCM HS/Results
Results
Objective 1: Sustainable inks by synthetic biology
To produce an orange ink using E. coli DH5α, we assemble in a pARK vector (α1), a transcriptional unit (TU) comprising a promoter, a ribosomal binding site (RBS), the gene codifying the enzyme flavanone3-hydroxylase (F3H), which is capable of catalyzing the transformation of naringenin into dihydrokaempferol, and a terminator. To check that the assembly of the TU within the vector is correct, we sequence the assembly. The result from sequencing can be checked in the parts registry. Additional proof of the correct assembly of this TU is obtained upon transforming the plasmid in E. coli DH5α. In particular, because an empty pARK1 (α1) vector provides bacteria with red colour, we can directly visualize successful clonings as white bacterial colonies, as shown in Fig. 1A. The efficiency of cloning the gene codifying F3H is particularly high as indicated by the large number of white colonies shown in the culture plate. Unfortunately, the efficiency is lower for cloning in pARK2 (α2) vector, and using the same procedure, the gene codifying the enzyme dihydro-flavonol 4-reductase (DRF), as shown in Fig. 1B. This DRF enzyme is required in the model anthocyanine route selected for this Project (see figure in the Design Section). The final validation necessary to confirm the success of this Level 1 cloning is obtained from the production of dihydrokaempferol. To do this, E. coli is cultured in LB broth in the presence of different concentrations of the colour precursor, naringenin, in concentrations ranging from 1 to 10 mM, as shown in Fig. 1C. Simply by eye, we observe that the eppendorf containing 10mM naringenin becomes orange upon culturing the bacteria, whereas in the lower concentrations of naringenin we only observe the characteristic yellow colour of the LB broth, as shown by the photographs of the eppendorfs in Fig. 1C. To better characterize this colour change, we measure the absorbance spectrum of the different conditions tested and compare them with that of a control. This control corresponds to raw LB broth containing 10 mM naringenin without any bacteria, and is shown by the black line in Fig. 1C. The red and green lines, which correspond to bacteria cultured with 1 and 2 mM naringenin, neither show any characteristic peak. However, an absorbance peak is obtained for bacteria cultured with 5 and 10 mM naringenin, as shown by the blue and cyan lines, respectively, in Fig. 1C.This is the prove that the enzyme flavanone3-hydroxylase (F3H) is catalyzing the transformation of naringenin into dihydrokaempferol. This is the major result of STAIN as it shows the successful cloning of a functional transcription unit and the synthetic transformation of bacteria to produce an orange pigment. Nevertheless, Level 2 assemblies will be required to complete the anthocyanine route used as a model in this project, which have not been successful yet, mainly as a question of time.
Fig. 1 A & B) Photographs of transformed E. coli DH5α showing different levels of efficiency in the cloning assembly of transcriptional unit (TU) in pARK vectors. A) Transformed DH5α where almost all colonies are white, exhibiting a high efficiency in the cloning of the transcriptional unit (TU) for the flavanone3-hydroxylase (F3H) gene. B) Transformed DH5α where most of the colonies are red, indicating a low level of efficiency in the assembly of the TU for the dihydro-flavonol 4-reductase (DRF) gene. C) In this panel it is represented the spectra of absorbance from 350nm to 750nm of cultures in LB media of transformed DH5α with vector pARK1 containing the TU for F3H, in presence of increasing amounts of precursor, 1mM, 2mM, 5mM and 10mM. The highest amount of naringenin exhibits the highest absorbance at 400nm. The inset shows a picture of these cultures where a deeper yellow is observed in presence of increasing amounts of precursor.
We have achieved cloning by Golden Gate strategy the TUs for the other three genes of the anthocyanin route (see figure in the Design Section), ANS for anthocyanidin synthase, 3GT for anthocyanin 3-O-glucosyltransferase and DFR for dihydroflavonol 4-reductase. ANS assembled in pARK1 (α1), and 3GT and DRF assembled in pARK2 (α2). Secuenciation proves the correct gene assembly in level 1 except for DRF that sequencing was not concluding. A double check is necessary before repeating the assembly. Though, the validation of these genes will be possible once we conclude level 2 assemblies to obtain coloured products that can be measured by spectrophotometry.
Objective 2: solving oxidation
Chemical Approach
To avoid oxidation of the biological inks that STAIN aims to produce, we characterize the tendency to be oxidized of different extracts obtained from different fruits and vegetables, and design an antioxidant system based on yeast. We work on these extracts for this part, instead of on the compounds obtained from our synthetic biology approach, because the compounds in the extracts are similar to those obtained in the synthetic biology approach and thus enables us to advance the two key objectives of the Project in parallel. With that in mind, the team extracts pigments from amaranth, red pepper, spinach, blueberries and red cabbage, some of them illustrated by the photographs shown in Fig. 2A. The extracts obtained can be lyophilized to preserve the colours in a solid state, as shown in Fig. 2B. In the extract obtained from red cabbage, changes in colour can be induced by pH changes. At almost neutral pH, the characteristic purple color of red cabbage is observed. However, an acidic pH turns the red cabbage extract red, whereas basic conditions turn it blue, as shown in Fig. 2C. The chemical reaction responsible for the colour change is the transformation of flavillium ions (red) into quinonoidal bases (blue). Importantly, this protonation reaction can be understood as well as an oxidation, and thus avoiding transformation of flavillium ions into quinonoidal bases can be used as an antioxidation strategy. Because the trasformation between flavilium ions and quinonoidal bases can be avoided by buffering the external media, we use citrate-phosphate buffers to avoid oxidation of these particular extracts. Liophylization of the buffered extracts thus results in colors that remain stable at least for the summer. These are shown in Fig. 2D, and constitutes another important result of STAIN.
Fig. 2 Representative photographs of the colors resulting from extracting and treating pigments from fruits and vegetables. A) From left to right: Photos of the team extracting pigments from amaranth, red cabbage and spinach. B) From left to right: Lyophilized extracts from red cabbage, strawberry, red pepper, amaranth, spinach and blueberry. C) Variation of colour induced by pH in red cabbage extracted pigments. The pH of the extracts is buffered using a citrate-phosphate buffer at pH 2.2, 3.2, 4.2, 5.2, 6.2, 7.2 and 8 from left to right. D) The different colours obtained upon varying pH are maintained upon lyophilization. The yellow colour is obtained by oxidation of the pigment extracted from red cabbage using hydrogen peroxide.
Besides by direct visualization, colours can be characterized by measuring the absorption spectra of each extract using a spectrophotometer. As an example, we show the spectra corresponding to red (R), blue (B) and green (G) colours obtained from red cabbage at pH 2.2, blueberries and spinach at neutral pH, respectively, in Fig. 3A. From the wavelength at which the maximum absorbance is observed, the colour of the extract can be predicted, owing to the complementarity between absorbance and transmission. For example, red cabbage at pH 2.2 exhibits a peak at 520 nm, which corresponds to green in the visible spectrum. If green light is absorbed, then red light is transmitted, which causes our eye to see such a red colour. Complementarily, we also measure the absorption spectra of the different colors obtained by changing the pH of the red cabbage extract, as shown in Fig. 3B. We observe that the wavelength of the maximum absorbance increases with pH in agreement with the color changes observed in the extracts, as shown in Fig. 2D.
Fig 3. Pigments characterization by spectrophotometry. Panel A) shows the spectra of extracted pigments of red cabbage at pH 2.2 in red, blue berry in blue and spinach in green the same day of extraction. Panel B) shows the spectra of red cabbage extracts at increasing pH.
Although red cabbage oxidation can be partially avoided by buffering the external environment, the mechanism by which other groups in the pigments are oxidized are independent on acid-base reactions and thus cannot be avoided through the use of buffers. For these cases, we design an alternative antioxidation strategy based on yeast. Yeast have a redox co-enzime composed by two nucleotides, nicotinamide and adenine, known as NADH in the reduced form and NAD+ in the oxidized form. If yeast cells are broken apart using a morter the NADH/NAD+ machinery can be liberated and used as a competitor for oxygen. We then mix the extracted pigments with the yeast cell-free system produced by these means to avoid oxidation of the extracted pigments. We demonstrate the efficiency of this antioxidant approach in a spinach extract as shown in Fig. 4, by mixing the extract with a 50 vol.% of yeast cell-free system. We observe that after 40 days there is a slight shift of two nanometers to the right in the maximum of absorbance for the pigment lacking the antioxidation treatment, whereas the maximum absorption is not shifted for the simple treated. Our results thus point to the potential of this design to avoid oxidation of natural inks, although the efficacy of the treatment should be tested for longer periods.
Figure 4. Spectra of spinach pigments with and without chemical antioxidation treatment. It is observed that after 40 days there is a slight shift of two nanometers to the right in the maximum of absorbance for the pigment lacking the antioxidation treatment.
Physical Approach
An alternative approach to avoid oxidation of extracts can be based on the creation of a physical barrier that avoids diffusion of oxigen into the extract. To do this, we propose to encapsulate extracts in oxigen-impermeable capsules using for example perfluorinated oils as shells because oxygen exhibits a very small solubility in perfluorinated compounds. Microfluidic technologies shows a great potential for the efficient encapsulation of substances. With this in mind, we build a glass-capillary microfluidic devices that enables the production of aqueous droplets containing the pigments extracted. This glass-capillary microfluidic device consists of a round capillary inserted into a square capillary as schematically illustrated in Fig. 5A. Within this device, the aqueous extracted pigment is injected through the left orifice of the square capillary, whereas an oil phase is injected through the interstices between the square and round capillaries, on the right. This configuration makes the two phases to get focused into the tip of the round capillary, in the center of the device, producing droplets of aqueous pigments that flow within the oil phase through the round collection capillary towards the exit. A photograph of the resultant glass-capillary device is shown in Fig. 5B. The resultant drops are collected in a vial, as shown in Fig. 5C. Microscope images of the central part of the device showing production of droplets and resultant drops in the vial are also included as Figs. 5D and 5E. For this particular experiment, the extracted pigment from red cabbage buffered at pH 2.2 was mixed with a 1wt.% CaCl2, on the idea to drop these droplets on a 2wt.% alginate solution to cause interfacial gelation of alginate on the surface of the drops, thus yielding alginate capsules. Unfortunately, we did not achieve alginate gelation using this approach. Further optimization of CaCl2/alginate ratios and of the protocol of transference of aqueous drops from oil to alginate is required next. Moreover, substitution of oil by a perfluorinated oil will be useful to avoid diffusion of oxygen into the encapsulated extract.
Fig. 5 A) Schematic illustration of the glass-capillary microfluidic device used to make drops of pigments. The aqueous pigment solution is flowed in opposite direction to a lipid containing-oil phase and the resultant drops are collected through a cylindrical capillary inserted at the right side of a square capillary. B) Photograph of the glass-capillary device on the microscope. C) Photograph of the drops of pigments collected in a vial. D) Microscope image showing microfluidic production of pigment drops running at typical flow rates of 1000 and 5000 μL/h for the aqueous and oil phases, respectively. E) Microscope image of the resulting production of pigment drops.
References
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