OVERVIEW
In fact, what our team does in the 2023 iGEM cycle will be very useful for future iGEM teams. Therefore, our work has been open and shared with the aim of maximizing the value of our work. In addition to doing solid work, our goal is to lay the foundation for future team expansion. Given the limited knowledge of synthetic biology in China, in addition to support education in remote areas, we donated to charities designed to support knowledge development in different youth groups.Our practice survey report on straw and non-grain feed can also well support the follow-up team projects.
Parts Collection
This year Wuhan-Hubei-China team's goal is to build restructuring wine yeast strains capable of degrading cellulose in the straw, and at the same time, by e. coli expression of xylanase efficient degradation of hemicellulose, high-protein fermentation production of straw feed.
Module 1: Design and construction of cellulase fusion gene
Firstly, we isolated β-glucosidase gene (BG) from sphingosphingomonas, exo-glucanase gene (CBH) from Reticulitermes flavomelus, and endo-glucanase gene (EG) from staphylostaphylus. The three selected enzymes had different sources, different classifications, simple structures and matching reaction conditions, which were consistent with the idea of fusion strategy.
| Code | BBa_K4795001 | BBa_K4795002 | BBa_K4795003 |
|---|---|---|---|
| Basic Part | EG | CBH | BG |
In order to achieve the expression of the triple functional cellulase fusion gene in Saccharomyces cerevisiae INVSc1, we used pHBM368-pgk, the same vector as the three separately expressed cellulases, as the vector of the expression system of saccharomyces cerevisiae. The vector is an expression vector of RDNA-mediated integration and has phosphoglycerate kinase (PGK1) promoter, α-factor signaling peptide, lactate deoxyriboside 5 '-phosphate decarboxylase gene (URA3) and ampicillin resistance gene.
Using the vectors pHBM368-pgk-BG, pHBM368-pgk-CBH and pHBM368-pgk-EG as templates, the catalytic domain CD coding sequences of cellulase BG, CBH and EG were obtained by PCR reaction. Primers are as follows:
Table note: The bold is the enzyme cutting site; The italic sequence is the reverse-coded sequence of the flexible joint (G4S) 3 (i.e. GGGGSGGGGSGGGGS).
The PCR products were recovered by ethanol precipitation and treated with the corresponding restriction enzyme. BG CD was cut with XbaⅠ enzyme, CBH CD was cut with XbaⅠ and EcoRⅠ enzyme, EG CD was cut with EcoRⅠ enzyme. The fragments were recovered by gel electrophoresis, and the fusion gene bce(BBa_K4795007) was obtained by T4 DNA ligase.
At the same time, the fusion gene bce fragment and the vector pHBM368-pgk-BG were treated with restriction enzymes SnaBⅠ and Eco81Ⅰ, and the DNA fragments were recovered by gel electrophoresis. The recovered fragments were mixed into the linking reaction system according to a certain mass concentration ratio (DNA fragments: linearized vector =10:1) to obtain the recombinant vector pHBM368-pgk-bce.
Module 2: Optimal mutation of xylanase gene
First, we isolated the xyn(BBa_K4795008) gene from Streptomyces sp.
Initially, in order to successfully express the xylanase gene in Saccharomyces cerevisiae EBY100, the vector PYD 1, containing the resistance gene AmpR, T7 promoter, T7 terminator, and green fluorescent protein (sf GF P), was used. However, because the target fragments was found to be difficult during the experiment, we abandoned this chassis and selected E. coli to express the target genes. And because xylanase expression in Escherichia coli will form inclusion bodies, affect the xylanase isolation purification, so we will promote sumo (BBa _ K4795009) and xylanase gene using Exnase II seamless connection, the target gene and the carrier with the same restriction enzyme digestion, and gel electrophoresis recovery of DNA fragments.
We use error-prone PCR for xylanase in vitro moleculars of directed evolution, by adjusting the proportion of dNTP, to xylanase randomly introduce mutation, build the mutation library, and finally screening and enzyme live test, improved xylanase gene, xyn 3-12 (BBa _ K4795014),the thirtieth position of the mutated xylanase translation sequence was changed from the original glycine to aspartate, xylanase SpecificActivity 29991.6U/mg, after mutation to 34804.9U/mg, increased by 16%.
Module 3: Use of promoter optimization to improve xylose isomerase activity
It was inspired by the previous study of replacing the inducible promoter with a strong component promoter to promote epidermal enzyme production, eliminating the limitations of saccharomyces cerevisiae induction. At the same time, the galactose induction system is inhibited by glucose, making it difficult to achieve a high level of expression. While the use of constitutive promoters is more stable than the inductor promoters, after consulting the data, we identified four constitutive promoters: ADH1(alcohol dehydrogenase), GAPDH(glyceraldehyde-3-phosphate dehydrogenase ), PDC1(pyruvate decarboxylase) and TEF1(translation extension factor).
| Code | BBa_K4795000 | BBa_K4795010 | BBa_K4795011 | BBa_K4795012 |
|---|---|---|---|---|
| Basic Part | ADH1 | GAPDH | PDC1 | TEF1 |
Xylose isomerase gene -- xylA(BBa_K47950013) was isolated from Orpinomyces sp. ukk1. The xylA gene fragment was cut with XbaI and NotI enzymes and cloned into the plasmid pHM368-pgk-cbh-ura cut with the same enzymes to obtain pHM368-PPGK-xylA-Ura. The promoters of ADH1, GAPDH, PDC1 and TEF1 were amplified by corresponding primers and inserted into pHM368-PPGK-xylA-Ura cut by NdeI and NotI by seamless and link-independent cloning. The expression plasmids pHM368-PADH1-xylA-Ura, pHM368-PGAPDH-xylA-Ura, pHM368-PPDC1-xylA-Ura and pHM368-PTEF1-xylA-Ura were constructed.
| Code | BBa_K4795017 | BBa_K4795018 | BBa_K4795019 | BBa_K4795020 |
|---|---|---|---|---|
| Composite Part | ADH1-xylA | GAPDH-xylA | PDC1-xylA | TEF1-xylA |
As can be seen from the picture, the XI activity of INVSc1/pHM368-PADH1-xylA reached 0.179 U at 48 h, which was 2.7-, 3.1- and 2.4-fold higher than INVSc1/pHM368-PPDC1-xylA (0.066 U), INVSc1/pHM368-PPGK-xylA (0.058 U) and INVSc1/pHM368-PTEF1-xylA (0.074 U), respectively. No XI activity was detected in INVSc1/pHM368-PGAPDH-xylA. XI activity of the engineered strains improved by promoter optimization strategy. According to this result, ADH1 promoter was the most efficient one for the expression of XI among these five promoters.
Effective education
Wuhan-Hubei-China has made great efforts in ensuring good education in synthetic biology, most importantly trying to increase overall participation in STEM teaching. In this sense, it is also important to share our learning and creation with the future iGEM teams and other teams, so that everyone can benefit from our work. Therefore, our animations, brochures and other documents are available for you to download. All of our popular science articles can also be viewed on our official wechat account HBERS iGEM.In addition, our initiated donations to charitable organized poor student programs can also contribute to the subsequent knowledge development of different youth groups.
Practice investigation on straw and feed
We have investigated and studied the treatment of straw, the cost, price and sales situation of breeding feed. The specific survey was conducted in the form of questionnaires and field visits, respectively. We have formed a detailed survey report for you to download and view, to provide data support for the subsequent team's two aspects of research.