Team:MADRID UCM HS/Design
Project design
How we made itOur goals and objectives
The scientific project STAIN (susSTAinable INk) focuses on the manufacture of natural and sustainable inks, or biological inks, which are not currently used due to their high oxidation tendency.
This project focused on two main objectives: The first one is to produce sustainable inks with synthetic biology, using as a model the metabolic route of the flavonoids and a precursor called Naringenin.
The second objective focuses on solving a problem that affects all organic inks, oxidation. With this, we try to maintain the original colors of the inks that we produce by slowing down, or ideally suppressing, the pigments' oxidation process by two main approaches, a chemical and a physical antioxidation.
Scheme of the project design
First objective: Synthesizing Inks
Our first objective consists of genetically modifying an E.coli to synthesize pigments starting from raw flavonoids as precursors. The Golden Gate Assembly strategy is used to assemble genes that codify for enzymes, flavanone3-hydroxylase, dihydroflavonol 4-reductase, anthocyanidin synthase and anthiocyanin 3-O-glucosylransferase in the route of anthocyanin synthesis that transforms naringenin (precursor) either into pelargonidin 3-O-glucoside or into kaempferol, producing color.
Fig. 1 Representation of the biosynthesis pathway of pelargonidin 3-O-glucoside from naringenin. The biosynthesis requires four genes: flavanone 3-hydroxylase (F3H) from Camelia sinensis (tea plant), anthocyanidin synthase (ANS) from Fragaria ananassa (strawberry), anthocyanin 3-O-glucosyltransferase (3GT) from Petunia hybrida (petunia) and dihydroflavonol 4-reductase (DFR) from Arabidopsis thaliana (arabidopsis). Enzyme names are in green together with the assembly level of the synthetic biology approach used for each step of the pathway. Bold dark blue arrows indicate the pelargonidin 3-O-glucoside biosynthesis pathway, whereas the dashed arrow indicates the route to the side-product kaempferol. Colored regions highlight changes in the naringenin molecule, resulting in color production.
Golden Gate assembly
The Golden Gate Assembly strategy enables the assembly of multiple DNA fragments in a vector in a single tube. This is possible by using the restriction enzyme BsaI-HFv2 to digest both the vector and the fragments through the recognition of asymmetric DNA sequences and cleavage outside the recognition sequences, and the T4 DNA ligase to assemble the fragments into the vector.
We propose an assembly divided into three phases, as shown in the diagram. Transcriptional units (TU) containing the genes are combined in pARK (α) vectors and 2 of these TUs are subsequently assembled in a new pDGB (Ω) plasmid. Lastly, the 4 TUs are cloned again in an pARK (α) vector. Combining the four different genes allows the cell to synthesize the four enzymes involved in the transformation of naringenin into pelargonidin 3-O-glucoside, thus changing its color.
Level 1
Consists of the assembly of a transcriptional unit (TU), composed of a promoter (P) BBa_K2656007, a ribosomal binding site (RBS) BBa_K2656009, the gene and a terminator (TT) BBa_K2656026 in a vector, using the Golden Gate Assembly strategy. Genes A and B are assembled in α 1 (pARK1) vector and genes C and D in α 2 (pARK2) vector. Using different vectors makes it possible to combine multiple transcriptional units. Only the plasmid containing TU A (F3H) BBa_K3255001 allows the cell to produce a light orange color. (See demonstrate page)
Fig. 2 A) Schematic illustration of the assembly of a transcriptional unit (TU), composed of promoter (P), a ribosomal binding site (RBS), a gene and a terminator (TT) in a vector using the Golden Gate Assembly strategy. The Golden Gate Assembly strategy enables the assembly of these four DNA fragments in a vector in a single tube using restriction enzyme BsaI-HFv2 to digest both the vector and the fragments through the recognition of asymmetric DNA sequences and cleavage outside of these recognition sequences, and the T4 DNA ligase to assemble the TU in the vector.
Level 2
Two transcriptional units (TU), each one containing a different gene, are assembled in a new vector using the Golden Gate Assembly strategy. Genes A and B are assembled in a new pDGB1 (Ω1) vector, while genes C and D are assembled in an pDGB2 (Ω2) vector.
Fig. 3 Schematic illustration of the assembly of two individual transcriptional units (TU), each one containing a different gene in a vector using the Golden Gate Assembly strategy. The Golden Gate Assembly strategy enables the assembly of these two TUs in a vector in a single tube using restriction enzyme BsaI-HFv2 to digest both the vector and the TUs through the recognition of asymmetric DNA sequences and cleavage outside of these recognition sequences, and the T4 DNA ligase to assemble the two TUs in the destination vector.
Level 2’
The final assembly phase consists of the assembly of four TUs, each one containing a different gene in a new pARK1 (α1) plasmid. Altogether, these four TUs allow the cell to produce a purple ink
Fig. 4 Schematic illustration of the assembly of four transcriptional units (TU), each one containing a different gene in a vector using the Golden Gate Assembly strategy. The Golden Gate Assembly strategy enables the assembly of these four TUs in a vector in a single tube using restriction enzyme BsaI-HFv2 to digest both the vector and the pairs of TUs, assembled previously in the pDGB (Ω) vectors, through the recognition of asymmetric DNA sequences and cleavage outside of these recognition sequences, and the T4 DNA ligase to assemble the two pairs of TUs in the destination vector.
Second objective: Stabilizing the pigments
Oxidation takes place when materials are exposed to an oxidizing agent, like oxygen. These agents change the pigments’ molecular structure, affecting their color. Therefore, one of our objectives is to maintain the original colors of the inks that we produce by slowing down, or ideally suppressing, the pigments’ oxidation process.
The time limitation made it necessary to create an experimental ink based on organic materials extracted from fruits, vegetables and leaves which contain molecules that behave in a similar way to the flavonoids synthesized by bacteria. Obtained pigments are initially characterized by using spectrophotometry. This technique gives information about the absorbance of the colors at different wavelengths and thus allows us to have an exhaustive control over the oxidation of the inks.
Our approaches to slow down the oxidation process and reinforce pigment stability include both physical and chemical antioxidation mechanisms.
A: From left to right: extracted pigments of amaranth, red cabbage and spinach.
B: Lyophilized extracts from red cabbage, strawberry, red pepper, amaranth, spinach and blueberry.
C: Red cabbage with different pH.
D: Lyophilized extracts from red cabbage with different pH and Hydrogen Peroxide.
Chemical antioxidation approach
The chemical antioxidation mechanism proposal is based on a cell-free system obtained from yeast cells. This system is created by breaking the membranes of the yeast cells leaving metabolites and its cellular machinery free. These metabolites such as NADH, tend to oxidize becoming NAD+ cation. Mixing this cell-free system with the pigments provokes a redox reaction where the metabolites oxidize, while the inks stay reduced, maintaining their original color for a longer period of time.
In a cell free yeast extract, free NADH oxidizes to cation NAD+ maintaining a reducing environment, thus avoiding the oxidation of flavillium cation to the quinonoidal base.
Physical antioxidation approach
The physical antioxidation approach focuses on creating a protective coating around the pigments using microfluidics, a groundbreaking technique in this area. This technique could help to isolate pigments from oxidizing agents thus maintaining a stable color.
A fluid current flow is created with pressure (mineral oil in the example) at the same time as the ink runs with a different pressure in the other direction. This makes the two fluids enter the thinner capillary which creates ink spheres while the other fluid (mineral oil) continues running out.
With the appropriate coating, the pigments should maintain their original color with accuracy and precision for a long time. This technique also produces versatile ink spheres that could be easily used in many different ways and industries.
