Team:BUCT-China/Design
Design
Our project is mainly divided into two sections, first section is the creation of bio-compatible material for scaffold, material that combined by polymer material of PHFA (poly hydroxyl fatty acid) and collagen. The second section is mixing animal cells with scaffold which is printed by 3D printer and culturing and differentiating cells to create cultured meat.
I. Section I
Our project is mainly divided into two sections, first section is the creation of bio-compatible material for scaffold, material that combined by polymer material of PHFA (poly hydroxyl fatty acid) and collagen. The second section is mixing animal cells with scaffold which is printed by 3D printer and culturing and differentiating cells to create cultured meat.
The first section can be achieved through four steps:
1. We take advantage of the fatty acid synthesis pathway of E.coli to synthesize myristoleic acid from glucose, we construct an artificial metabolic pathway that can desaturate the ACP-fatty acid and finish the fatty acid synthesis to produce 14C fatty acid.
2. We use another E.coli as chassis bacterial to hydroxylate myristoleic acid from step1 as hydroxy myristoleic acid, the genes of fatty acid transferase and hydroxylase are transferred into bacteria.
3. Based on synthetic biology, we constructed another engineered bacterium to utilize hydroxy myristoleic acid to synthesize product the new material PHFA(poly hydroxyl fatty acid) through polymerization of hydroxy fatty acid.
4. We also using plasmids as vector to synthesize functional collagen in prokaryotic E.coli, collagen as an ingredient of scaffold can not only improve the texture of cultured meat, but also simulating the surrounding environment of growing cells.
By combination of PHFA and collagen as a raw material of 3D printing scaffold.
Step 1 Synthesis of Mystoleic Acid
In this step we will synthesize the precursor of PHFA monomer using glucose, as an attempt to propose the choice of medium and long chain fatty acids as the precursor of the monomer, we know that long chain fatty acids have low solubility and short chain fatty acids may not hard enough as scaffold material. Therefore, we expected to choose medium-length chain, which would be stronger than short-chain fatty acids and would have higher solubility than long-chain fatty acids. In addition, it has been proven that a reasonable proportion of polyunsaturated fatty acids in the diet has been found to be effective in maintaining good health, promoting growth and development, reducing the incidence of cardiovascular diseases, lowering blood lipids and blood pressure, immunomodulatory, anti-inflammatory, and anti-cancer effects. [1] Therefore, we chose medium and long chain unsaturated fatty acids as the raw material for cell growth scaffolds. In experimental design, we passed the raw material myristoleic acid by expressing desaturase and thioesterase for cultured meat cell culture scaffold, and we decided to express ACP-desaturase TsFab2 and thioesterase BTE, respectively, in E. coli, and finally achieved the synthesis of myristoleic acid in E. coli.
We chose ACP-desaturase as the desaturase that catalyzes the desaturation of fatty acid-ACP to form a double bond by dehydrogenation between the 9th and 10th carbon atoms, and acyl carrier protein (ACP) desaturase is a key enzyme in fatty acid anabolism. The double bond is composed only in the acyl carrier protein bound state of fatty acids, which are found mainly in the cytoplasm in soluble form. The specific substrate, fatty acid-ACP molecule, performs a redox reaction of the 9th and 10th carbon atoms of the fatty acid carbon chain by binding to the enzyme active center located inside the groove on the surface of the dimer molecule. This is the first step in the desaturating enzyme of the typical plant unsaturated fatty acid biosynthetic pathway, which directly regulates the ratio of saturated to unsaturated fatty acids in membrane and storage lipids. The stearoyl-acyl carrier protein desaturase gene will change the content of saturated versus unsaturated fatty acids in fatty acids[2]. Δ9 desaturase catalyzes the formation of the first double bond in fatty acids and is a key regulatory enzyme in the biosynthesis of unsaturated fatty acids, and in cyanobacteria it is also known as DesC. Its function is to catalyze the conversion of 16:0-ACP and 18:0-ACP to 16:1(Δ9)-ACP and 18:1(Δ9)-ACP [4]. In addition, Nishizaki[5]also cloned the Δ9 desaturase gene DesC from A. nidulans, which can interact with C16 and C18 fatty acids of various membrane lipids to form Δ9-site double bonds. Our group selected Chlamydomonas reinhardtii CC503 FAB2, a plastidial acyl-acyl carrier protein fatty acid desaturase that can convert 18:0-ACP to 18:1(Δ9)-ACP, adding a double bond in the biosynthetic pathway of polyunsaturated fatty acids, and Triadica sebifera stearoyl-ACP desaturase from Sapium sebiferum was used as the desaturase enzyme needed in the experiment, and parallel experiments were performed with the two desaturases to test the experimental results and select the enzyme with excellent endogenous expression as the final enzyme for the experiment.
Thioesterase BTE was chosen as the enzyme that catalyzes the termination of unsaturated fatty acid chains, hydrolyzes the thioester bonds of acyl-S-acyl carrier proteins, and releases the fatty acids. The thioesterase BTE from California laurel is already available in the laboratory and was chosen to ensure the efficiency and success of the experiment. In addition, the literature shows that BTE is more effective than other thioesterase in relieving the feedback inhibition of the E. coli fatty acid de novo synthesis pathway by lipoyl- ACP, thus promoting a significant accumulation of medium carbon fatty acids (C12 and C14).[6] Moreover, the BTE exhibited high substrate catalytic properties for C12 chain-length lipoyl-ACP. [7] Meanwhile, the recombinant strain is able to efficiently convert cheap glucose to generate short-chain fatty acids, and this conversion process can be carried out under mild conditions (30-37°C) with good product specificity, which effectively reduces the production cost and is a good method for short-chain fatty acid synthesis. [8] The results showed that the intracellular free fatty acid content was increased by heterologous expression of BTE; meanwhile, the expression of thioesterase BTE also changed the chain length composition of free fatty acids, and the chain length of free fatty acids produced were mainly C12 and C14, which accounted for more than 80% of the total fatty acid production, reaching 118.9 mg/L, and more than 95% of them were present in the cells. [9] Therefore, we selected California lauryl thioesterase BTE as the thioesterase used in the recombinant plasmid.
At the same time, there is a problem in this reaction process. We found that in the E. coli type-II fatty acid synthesis pathway, thioesterase is responsible for catalyzing the hydrolysis of acyl-ACP releasing free fatty acids, while ACP desaturase have to catalyze the same substrate fatty acid-ACP to produce desaturated fatty aicd- ACP, if both enzymes were endogenously expressed simultaneously in the E. coli type-II fatty acid synthesis pathway, both thioesterase and ACP desaturase would act simultaneously on the fatty acids produced in the type-II fatty acid synthesis pathway. This would prevent the synthesis of our desired desaturated medium and long chain fatty acids and would result in substrate competition. To solve this problem, we used operon to achieve regulation of gene expression by modifying the expressed enzymes using arabinose and lactose operon. We combined the arabinose operon with the ACP desaturase TsFab2 and the lactose operon with the thioesterase BTE in order to achieve the synthesis of ester-ACP by first expressing the ACP desaturase gene in the E. coli fatty acid synthesis pathway, and then expressing a certain amount of ester-ACP in E. coli by regulating the lactose operon, the thioesterase BTE expression was made to release the feedback inhibition of ester-ACP and convert the synthesized ester-ACP into the target product myristoleic acid.
Step 2 Hydroxylation
In Hydroxylation, we designed and constructed a plasmid containing FadL gene and P450 BM3 gene sequences. After the plasmid was introduced into the FadD gene-deficient E. coli cells in the laboratory, the cells achieved increased absorption of exogenous long-chain fatty acids (LCFA). Then, hydroxylation reaction was carried out to make the host cells produce more hydroxyl fatty acids.
The outer membrane protein FadL of Escherichia coli is a necessary carrier for transporting exogenous fatty acids, which helps LCFA to cross the outer membrane, especially for transporting fatty acids with 10-18 carbons [10] [11] .However, there is also a FadD gene in Escherichia coli, which can catalyze the synthesis of long-chain fatty acyl-CoA (LCFA-CoA), and then participate in β-oxidation and be consumed under the catalysis of FadE. The specific process is shown in Fig.2, in which FadR gene plays a regulatory role in the whole process [12].
Our objective was to hydroxylate LCFA which had already entered into the membrane. Thus, the presence of the FadD gene would hinder the experiment. By consulting the relevant literature, we found that Jin H and his team have done two experiments to prove that knockout of FadD gene and overexpression of Fadl in Escherichia coli can increase the production of hydroxylate in Escherichia coli. First, they knocked out the FadD gene in E. coli and introduced the P450 enzyme gene into the bacteria to convert palmitic acid into omega-hydroxypalmitic acid. The results of the experiments demonstrate that the production of ω-hydroxypalmitic acid is increased in the FadD-deleted, P450 overexpressed mutants. The experimental results are shown in fig. 3 (a). [12]
Later, they overexpressed Fadl gene on the basis of FadD gene deletion and P450 expression and found that the production of ω-hydroxypalmitic acid was greatly increased. The experimental results are shown in fig. 3 (B). Because LCFA accumulates in the periplasm through FadL membrane protein transport and is consumed in the cytoplasm through P450 enzyme, the diffusion of LCFA is driven and will not be participated in β-Oxidation. As a result, the yield of hydroxylation products increases. [12]
The P450 enzymes mentioned in the experiment are heme-containing monooxygenases. Although different CYP450 species require different electron transport systems, their catalytic reactions can generally be represented by the following general formula:
P450 BM3 is a particularly active member of the P450 monooxygenase family, which catalyzes the hydroxylation of ω-1, ω-2 and ω-3 positions of monounsaturated fatty acids, fatty alcohols and fatty amides with a chain length of C12 ~ C20. [12] [14] It not only conforms to the type of hydroxylase used in the literature, but also has stronger oxidation ability. The catalytic substrate is LCFA, and the expression content in Escherichia coli is high [12] [14], so we choose it as our oxidase.
Step 3 Polymerization
In step3 we are using two gene sequences: EC (long chain acyl CoA synthase) and WS2 (wax ester synthase).It could express two proteins, CoA ligase and acyltransferase. The function of the two proteins as follows (EC for CoA ligase; WS2 for acyltransferase) At the same time, in our design, it contained a T7 promoter of EC between the two segments (EC / WS2)
In order to verify the enzymes’ functions, we constructed the plasmid pETDuet (WS2/EC). In the verification experiment, we used hydroxydodecanoic acid and nonanoic acid as substrates. We expressed pETDuet (WS2/EC) into E. coli BL21 (DE3). The functions of EC and WS2 are shown below. During the polymerization process, EC acts as a CoA ligase to remove H in CoA-SH and -OH in the carboxyl group of hydroxy fatty acid through dehydration and condensation, and connect CoA and hydroxy fatty acid at the same time; WS2 As an acyltransferase, the acyl group in the hydroxy fatty acid CoA replaces the H in hydroxyl group of the hydroxy fatty acid, and CoA-SH is generated at the same time.
We set 1 group of control substrates nonanoic acid and hydroxydodecanoic acid and 3 groups of experimental group substrates were 1. Dodecanoic acid and isopropanol (blue line) 2. Nonanoic acid and hydroxydodecanoic acid (purple line) 3. Nonanoic acid and isopropanol (black line). By adding 3~4% DMSO to increase the solubility of the substrate hydroxy fatty acid and the permeability of the cell membrane, after constructing the engineered bacteria, protein expression and induction fermentation are carried out. The fermentation product was extracted with chloroform for further gas phase product analysis. The experiments are designed to use hydroxydodecanoic acid instead of hydroxy myristoleic acid is taking consideration of solubility of middle-long chain hydroxy fatty acid. The shorter length of chain, the better the solubility. Besides, hydroxydodecanoic acid is also a qualify monomer. Using 12C hydroxy fatty acid can avoid the potential impediment that lacks of sufficient substrate for polymerization in solute phase, so that we choose to use hydroxydodecanoic acid to testing the feasibility of our polymerization pathway.
Step 4 Synthesis of Collagen
Our experimental group planned to provide a better cell culture environment for artificial meat by expressing hydroxylated collagen. We selected the expression of collagen hCOL3 and L-593 proline hydroxylase (Hydroxylated proline can significantly improve the anti-degradation ability of peptides.[13]) in E.coli (BL21) respectively(considering the stability and activity of expression in E. coli), then carried out enzyme modification reaction in suitable and vitro environment, finally, purified the hydroxylated collagen and i determined its content.[14]
We planned to use bacterially active prolyl hydroxylase enzymes from the giant virus mimivirus to produce hydroxylated collagen.[15] The L593 gene was amplified by primers, and the expression vector without his tag was prepared. The corresponding amplifiers were introduced into pET16b, and L593 was linearized with Ncole-BamHL and pET16b-L593.For figure 2, it is the pET16b-L593 vector that we synthesized uses pET-16b as the expression vector and lacks His-Tag.
5’-CCATGGATGAAAACTGTAACAATAATCACG-3’
5’-CGAACGTAAGTTCAGCTAAGGATCC-3’
We chose to use a fragment of human collagen type III COL3A1cDNA(hCOL3), encompassing 1206 bp and lacking propeptide-encoding region. The pET28a expression vector was digested with Ncol-BamHI to eliminate the His tag at the 5N terminal. The obtained hCOL3 fragment was inserted into pET28a as a Ncol-BamHI fragment to get pET28a-hCOL3 with His-tag.
NcoI 5’-CCATGGATGTACGACAGTTATG-3’
BamHI 5’-CCACCATCACCACTAAGGATCC-3’
Experimental operation:
Escherichia coli as a chassis organism expressed collagen (35.8KDa+840.9dADa) + L593 (27.9kDa) respectively, and explored the optimal conditions at 30 degrees Celsius and 18 degrees Celsius.[16] We also modified the successfully expressed hCOL3 protein with L593 hydroxylase in a suitable in vitro environment to obtain hydroxylated collagen. Because it has His-tag, it can be purified. Collagen was purified by Ni column, SDS electrophoresis and Coomassie brilliant blue method was used to measure the protein content (expressed at 30 degrees Celsius and 18 degrees Celsius).
II. Section II
In the second section, we select chicken muscle satellite cells as seed cells mixed with scaffolds for co-printing. we create various 3D model on software with diverse percentage of materials to print ideal scaffold. To better understand the cell growing process and give a support for future studies, we establish a mathematics model to simulate the growing of cultured meat.
The manufacture of biological scaffolds is one of the important contents in the field of tissue engineering research. The ideal biological scaffolds must have good biocompatibility, meet the conditions of cell growth, not reject the human body, have certain mechanical properties, and can be degraded. At the same time, the application of 3D printing technology in the field of tissue engineering provides a new idea for the manufacture of tissue engineering scaffold, which has the advantages of fast and accurate.
We find that sodium alginate/gelatin composite scaffolds have received widespread attention and have good 3D molding ability. Sodium alginate has chemical cross-linking with divalent ions to obtain stable characteristics, gelatin is better at low temperature molding, can be used in the printing of chemical cross-linking + low temperature platform method to obtain stable base support. So, our team decided to use sodium alginate/gelatin as the raw material for the printing base scaffold.
PHFA can easily bind the protein ligand of cell membrane receptor to stimulate cell adhesion and growth, which is a very good tissue engineering material. Collagen is rich in cell attachment sites and can better simulate the external growth environment of muscle cells. At the same time, it has a strong ability to adsorb water molecules. Abundant water environment can protect cells and provide a good channel for the transport of nutrients needed by cells and the discharge of metabolites. After the printing of the basic scaffold, PHFA and collagen were added to improve the overall performance of the mixed material and make it more conducive to cell growth.
In this study, we make gelatin-sodium alginate composite hydrogels of different proportions by adding sodium alginate into the gelatin solution, and evaluate the basic properties such as molding, stability and porosity of printed scaffolds of different proportions. After determining the optimal ratio, PHFA and collagen solution are added to the scaffold and mixed with the cells to print, to evaluate the growth effect of the final cells on the scaffold.
In order to produce cultured meat through tissue engineering knowledge, cell seeds are also a very important factor in addition to the biological scaffold material produced by the first three groups.
Our team selected chicken muscle satellite cells as seed cells mixed with scaffolds for co-printing. Chicken-derived muscle satellite cells are a type of stem cell that has the potential to differentiate into muscle, which is high in protein. When these cells are attached to the PHFA and collagen composite scaffold, they can differentiate into the nutrient-rich "cultured meat" that we need. The presence of collagen scaffolders also gives cultured meat a richer taste.
Since chicken-derived muscle satellite cells are derived from chicken embryos rather than live chickens, they provide better animal welfare. More importantly, chicken has no religious restrictions and is higher in protein, more delicate and digestible, and more widely available.
1．Basic scaffold experiment
1.1 Preparation of scaffold experimental:
Experimental materials: Sodium alginate powder, gelatin particles, calcium chloride particles ，PBS (to maintain PH stability of solution, suitable for cell growth) Experimental device: The whole printing process is completed by extruded 3D printing equipment built by ourselves. The whole equipment is composed of control system, motion mechanism, feeding system and sprinkler system
1.2 Scaffold experimental operation:
1. Under the condition of 70℃ water bath heating, stirring for 2h, the gelatin-sodium alginate composite hydrogel solution is prepared by gelatin concentrations of 10%, 15%, 20%, 25% (W/V) and sodium alginate concentrations of 3%, 5%, 6% (W/V) . The solution is put into ultraviolet disinfection cabinet for 24h disinfection to ensure sterility. Anhydrous calcium chloride particles are prepared with deionized water into a 4% (W/V) solution and sterilized by the same sterilization method.
2. Rheological test: The rheological properties of gelatin-sodium alginate composite hydrogel are measured by laboratory biomaterial rheometer. The curves are obtained by measuring the viscosity of materials in different proportions by varying the shear rate.
3. G code design: draw the required 3D models with CAD and import them into Slic3r (a slicing software) for slicing and printing path planning, and set printer parameters, such as model size, height, filling rate, etc. Use different parameters to plan different printing paths and generate corresponding G code files.
4. Printing experiment: Fill the prepared material into the bio-ink cartridge and install it into the 3D printer. Open the air pressure pump, adjust the distance between the nozzle position and the low-temperature platform, set the initial air pressure and temperature for printing, and constantly adjust the parameter values in the printing process to obtain a well-shaped scaffold. After printing, take the scaffold quickly, add 4% (W/V) anhydrous calcium chloride for half an hour, take it out and put it into the orifice plate, and store it at 4℃.
5. Characterization of porosity: We measure the porosity of the scaffold sample by using the ethanol extraction method. First, weigh the weight W1 of the pycnometer filled with ethanol and Ws of the dried sample, then weigh the weight W2 of the pycnometer filled with anhydrous ethanol after the fully saturated ethanol scaffold, and finally weigh the weight W3 of the pycnometer removed from the scaffold.
Due to the sample volume
6. Lyophilization of the scaffold: Put the printed scaffold into the six-hole plate, cover it with plastic wrap and puncture holes, and put it into the lyophilization machine for 2 days
7. Stability test of the scaffold: Put the printed scaffold into the well plate, add the cell culture medium until it was completely covered by the scaffold, and place the scaffold under the condition of cell culture for 5 days to observe the morphological changes of the scaffold.
2.Cells experiment
2.1 Cell experimental materials:
Raw material: 12-day-old chicken embryo
Muscle tissue was extracted from 12-day-old chicken embryos. The most important reason was that chicken embryos had more muscle satellite cells and were more active. The second reason was that chicken embryos had not yet developed into real chickens and could be regarded as eggs, which was beneficial to animal welfare to some extent. Some vegetarians who can eat eggs can also eat our cultured meat.
Complete medium: DMEM-F12 medium (Gibco)+ fetal bovine serum (Gibco) 15% + penicillin-streptomycin 2%
Mixed enzyme solution: 20% collagenase + 10% trypsin +PBS
Differentiation medium: DMEM-F12 medium (Gibco)+ horse serum (Gibco) 4% + penicillin-streptomycin 2%
Cell staining：Neutral Red Staining Solution
2.2 Cell experimental operation:
1. Primary extraction of chicken muscle satellite cells
The legs below the hip joint of the chicken embryo were removed with a scalpel of strict disinfection and sterilization, and non-muscle tissues such as fascia, fat and bone were removed. The muscle tissue was cut into small pieces and placed in 75vol % ethanol solution for 1 min to prevent contamination. Then, the muscle tissue was washed with PBS buffer 3 times to remove the blood clots, and the muscle was cut into minced meat shape with curved eye scissors. Mixed enzyme solution twice the volume of the tissue was added, and the tissue was placed in a 37℃ constant temperature gas bath shaker for digestion for 30min, upside down every 10 minutes. After digestion, the same volume of complete medium was added to terminate digestion. The digestive liquid was passed through 100 mesh and 200 mesh cell filter to obtain cell suspension. Then the cells were placed in a centrifuge for 1500r, 10min, and centrifuged to remove the supernatant and obtain cell precipitation. The cell precipitated was suspended in the complete culture medium, and then transferred to the culture flask, which was incubated at 37℃ for 30min. The nonadherent cell suspension was transferred to the new culture flask for secondary differential adherent, so as to remove the fibroblasts. After 48h, the solution was changed and the tissue fragments, dead cells and non-adherable red blood cells in the culture medium were removed to complete the purification.
2. Culture and passage of chicken muscle satellite cells
When the cells grow to cover 80% of the flask area, the passage operation can be carried out. The original medium was first sucked out, then 1ml trypsin was added to the culture flask, digested in the incubator for 1 min, and 3ml complete medium was added to terminate digestion. The pipette gun was used to produce uniform cell suspensions, and then the cell suspensions were averaged into two new culture bottles to complete cell passage.
3. 3D co-printing of composite scaffolds and cells
3.1 Experimental materials:
PHFA and collagen solution, PBS，Calcein-AM/PI Double Stain Kit3.2 Co-printing experimental operation：
It has been found through previous study that the mixture of sodium alginate/gelatin with a ratio of 3:25 is the most suitable condition for the basic scaffold. We believe that the appropriate cell inoculation density is 10^ 6 cells per 1ml, which can not only evenly distribute cells on the scaffold, but also meet the needs of subsequent cell growth and differentiation.
Take the sodium alginate/gelatin raw material that has been destroyed in advance and mix it together in a ratio of 3:25 according to the previous method. P3 muscle satellite cells were removed from the incubator, digested from the wall of the culture flask with trypsin and gently blown to prepare cell suspension, followed by cell counting, and the final cell suspension concentration was 1ml of 10^ 6 cells.
After the basic scaffold was mixed evenly, 1ml cell suspension and PHFA and collagen solution prepared in advance were added and mixed evenly with the basic scaffold. Then 3D printing was carried out according to the printing mode and conditions of the basic scaffold.
The printed mixed scaffold will be immediately fixed in a calcium chloride solution and then transferred to a well plate with DMEM-F12 medium and incubated in an incubator for one day.
After one day of co-culture, cell activity was determined by neutral red staining to determine the number of viable cells. By adding neutral red dye solution to stain, the cells will not be affected, the cells can continue to survive, but also can be more obvious to indicate the cells.
What's more, the Calcein-AM/PI Double Stain Kit will be used in testing the number of live and dead cells. We made 2D and 3D image display.
Mathematic model
For detail please go to https://2021.igem.org/Team:BUCT-China/Model
References
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